Method for engineering immunoglobulins

ABSTRACT

The present invention relates to a method for engineering an immunoglobulin comprising a variable domain and at least one modification in at least two structural loops of said immunoglobulin and determining the binding of said immunoglobulin to an epitope of an antigen, wherein the unmodified immunoglobulin does not significantly bind to said epitope, comprising the steps of: providing a nucleic acid encoding an immunoglobulin comprising at least two structural loops, modifying at least one nucleotide residue of each of said structural loops, transferring said modified nucleic acid in an expression System, expressing said modified immunoglobulin, contacting the expressed modified immunoglobulin with an epitope, and determining whether said modified immunoglobulin binds to said epitope, immunoglobulins produced by such a method and libraries of immunoglobulins.

This application is a continuation of U.S. application Ser. No.14/853,919, filed Sep. 14, 2015, which is a continuation of U.S.application Ser. No. 12/307,569, filed Sep. 21, 2009, which is a 371national phase filing of PCT/AT2007/000343, filed Jul. 5, 2007, whichclaims the benefit of Austria A 1145/2006, filed Jul. 5, 2006, thecontents of each of which are incorporated herein by reference in theirentirety.

FIELD OF INVENTION

The present invention relates to a method for engineering andmanufacturing of a molecule comprising a modified immunoglobulinvariable domain polypeptide. The general field is the engineering ofproteins with the aim to impart them with specific binding properties.More specifically, the engineered proteins of relevance here areimmunoglobulins (antibodies), and even more specifically, singlevariable domains or pairs or combinations of single variable domains ofimmunoglobulins or combinations with other immunoglobulin domain˜. Thespecific binding properties of immunoglobulins are important featuressince they control the interaction with other molecules such asantigens, and render immunoglobulins useful for diagnostic andtherapeutic applications.

BACKGROUND Structure of Antibodies

The basic structure of antibodies is similar for various classes ofantibodies and for various species and will be explained here using asexample an intact IgG1 immunoglobulin. Two identical heavy (H) and twoidentical light (L) chains combine to form the Y-shaped antibodymolecule. The heavy chains each have four domains. The amino terminalvariable domains (VH) are at the tips of the Y. These are followed bythree constant domains: CH1, CH2, and the carboxy-terminal CH3, at thebase of the Y's stem. A short stretch, the switch, connects the heavychain variable and constant regions. The hinge connects CH2 and CH3 (theFe fragment) to the remainder of the antibody (the Fab fragments). OneFe and two identical Fab fragments can be produced by proteolyticcleavage of the hinge in an intact antibody molecule. The light chainsare constructed of two domains, variable (VL) and constant (CL),separated by a switch.

Disulfide bonds in the hinge region connect the two heavy chains. Thelight chains are coupled to the heavy chains by additional disulfidebonds.

The variable regions of both the heavy and light chains (VH) and (VL)lie at the “tips” of the Y, where they are positioned to react withantigen. This tip of the molecule is the side on which the N-termini ofthe amino acid sequences are located.

The stem of the Y projects in a way to efficiently mediate effectorfunctions such as the activation of complement and interaction with Fcreceptors, or ADCC and ADCP. Its CH2 and CH3 domains bulge to facilitateinteraction with effector proteins. The C-terminus of the amino acidsequence is located on the opposite side of the tip, which can be termed“bottom” of the Y.

Immunoglobulin Variable Domains (V Domains)

Each domain in an antibody molecule has a similar structure of two betasheets packed tightly against each other in a compressed antiparallelbeta barrel. This conserved structure is termed the immunoglobulin fold.For reference see Bork et al. (1994) J. Mol. Biol. 242:309-320; Halabyet al. (1999) Protein Engineering 12: 563-571; Immunobiology. 5th ed.Janeway, Charles A.; Travers, Paul; Walport, Mark; Shlomchik, Mark. NewYork and London: Garland Publishing; 2001.

The fold of variable domains has 9 beta strands arranged in two sheetsof 4 and 5 strands. The 5-stranded sheet is structurally homologous tothe 3-stranded sheet of constant domains, but contains the extra strandsC′ and C″. The remainder of the strands (A, B, C, D, E, F, G) have thesame topology and similar structure as their counterparts in constantdomain immunoglobulin folds. A disulfide bond links strands B and F inopposite sheets, as in constant domains. The variable domains of bothlight and heavy immunoglobulin chains contain three hypervariable loops,or complementarity-determining regions (CDRs). The three CDRs of a Vdomain (CDR1, CDR2, CDR3) cluster at one end of the beta barrel. TheCDRs are loops that connect beta strands B-C, C′-C″, and F-G of theimmunoglobulin fold. The residues in the CDRs vary from oneimmunoglobulin molecule to the next, imparting antigen specificity toeach antibody.

The VL and VH domains at the tips of antibody molecules are closelypacked such that the 6 CDRs (3 on each domain) cooperate in constructinga surface (or cavity) for antigen-specific binding. The natural antigenbinding site of an antibody thus is composed of the loops which connectstrands B-C, C′-C″, and F-G of the light chain variable domain andstrands B-C, C′-C″, and F-G of the heavy chain variable domain.

Scaffolds for Protein Engineering

Using the 3D structure of a protein as an aid for design, amino acidresidues located on the surface of many proteins have been randomizedusing the core structure of the protein as scaffold. Examples for thisstrategy are described or reviewed in the following references andincorporated herein by reference: Nygren P A, Uhlen M., Curr Opin StructBiol. (1997) 7:463-9; Binz H K, Amstutz P, Kohl A, Stumpp M T, Briand C,Forrer P, Grutter M G, Pluckthun A. Nat Biotechnol. (2004) 22:575-82;Vogt M, Skerra A. Chembiochem. (2004) 5:191-9; U.S. Pat. No. 6,562,617;Hufton et al. FEBS Letters (2000) 475: 225; Binz et al. Nat Biotechnol.(2005) 23:1257-68; Hosse et al. Protein Sci. (2006)15:14-27.

The basic principle of this technique is based on the observation thatmany proteins have a stable core, formed by specific arrangements ofsecondary structure elements such as beta sheets or alpha helices, whichare interconnected by structures such as loops, turns, or random coils.Typically, these latter three structure elements are less crucial forthe overall structure of the protein, and amino acid residues in thesestructure elements can be exchanged often without destroying the generalfold of the protein. Naturally occurring examples for this designprinciple are the CDRs of immunoglobulin-like domains as can be found inantibodies, T-cell receptors and other molecules of the immunoglobulinsuperfamily. Artificial examples include lipocalins, ankyrins, kunitzdomain inhibitor, knottin and other protein scaffolds.

Manipulation of the CDR-Loops of Immunoglobulin Variable Domains

A multitude of prior art documents show that the immunoglobulin likescaffold has been employed for the purpose of manipulating the existingantigen- or ligand-binding site, thereby introducing novel bindingproperties. More precisely, mainly the CDR regions have been engineeredfor antigen binding, in other words, in the case of the immunoglobulinfold, the natural antigen binding site has been modified in order tochange its binding affinity or specificity. A vast body of literatureexist which describes different formats of such manipulatedimmunoglobulins, frequently expressed in the form of completeantibodies, fusion products and/or fragments such as single-chain Fvfragments (scFv), diabodies, minibodies, single domains or Fab fragmentsand the like, either displayed on the surface of phage particles orother viruses and cells or solubly expressed in various prokaryotic oreukaryotic expression systems. The techniques are reviewed e.g. inHolliger & Hudson, Nat. Biotechnol. (2005) 23:1126-36 and Hoogenboom,Nat. Biotechnol. (2005)23:1105-16.

Framework or Non-CDR-Regions of Immunoglobulin Variable Domains

The CDR-loops of an immunoglobulin variable domain define the antigenspecificity. The rest of the molecule is termed framework (FR). Theseframework regions however are composed of beta-strand and loopstructures.

The loops which are not CDR-loops in a native immunoglobulin variabledomain do not have antigen-binding or epitope-binding specificity butcontribute to the correct overall folding of the immunoglobulin domainand consequently also to the correct positioning of the CDRs and tointeraction between domains. These loops are named structural loops forthe purpose of this invention.

Antibody variable domains in general have been manipulated for very manydifferent reasons, such as the construction of various antibody formats,CDR grafting (i.e. grafting of specificity of a particular antibody intoanother framework; e.g. Jones et al. Nature (1986) 321: 522-525;Kashmiri et al. Methods (2005) 36:25-34), changing the surface ofvariable domains in order to make it more soluble and stable (e.g. Ewertet al. Methods (2004) 34:184-99; Conrath et al. J Mol Biol. (2005)350:112-125), to render it monomeric (e.g. Dottorini et al. Biochemistry(2004) 43:622-628))or to study the interaction between variable domains(e.g. Masuda et al. FEBS J. (2006) 273:2184-94). Many of thosemanipulations have involved changes in the framework region of themolecule; some amino acid mutations within structural loops of thevariable domain have been performed.

The influence of remote framework regions on CDR-loop positioning isevident from CDR grafting results which show that mutation of frameworkamino acids frequently is necessary to regain antigen binding aftergrafting of CDRs from one framework to another (e.g. Foote & Winter(1992) J. Mol. Biol. 224: 487-499; Kettleborough et al. Protein Eng.(1991) 4:773-783; Wu et al. J. Mol. Biol. (1999) 294:151-162).

Simon & Rajwesky (Protein Sci. (1992) 5:229-234) have studied theeffects of a four residue insertion into the FR3 loop of the heavy chainvariable region from the anti-NP antibody B1-8. The insertion mutant wasobtained as secreted antibody without major defects in biosynthesis,indicating that antibody variable domains can accommodate lengthvariation not only in complementarity determining regions (CDRs), butalso in framework region (FR) loops. In this case the original antigenbinding site formed by the CDR-loops was not affected by themodification in a neighbouring structural loop.

Grafting of CDR-Loops into Structural Loop Regions

EP0640130B1 describes chimeric immunoglobulin superfamily proteinanalogues (chi-proteins) having more than one biological binding site(single V domains or Fvs). The binding sites on these proteins arecomprised of hypervariable regions derived from molecules related to theimmunoglobulin superfamily of molecules, including immunoglobulins, cellsurface antigens (such as T-cell antigens) and cell receptors (such asFc-receptor). The hypervariable regions are called “CDR-like regions”and define ligand binding sites. Additionally, the chi-protein has atleast one more ligand binding site segment, also a CDR-like region,spliced into the FR-like regions of the beta-barrel domain.

Each ligand binding site of the chi-protein therefore is comprised of aCDR-like region derived from molecules of the immunoglobulinsuperfamily. For example, a ligand binding site is comprised of the CDRsderived from an immunoglobulin molecule whose ligand is an antigen.

EP0640130B1 thus teaches how to splice CDR-like regions with a givenspecificity from a molecule of the immunoglobulin superfamily into thestructural loops of a variable domain. It is postulated that functionalbispecific antibodies can be prepared by this technique. There is arequirement for this technique that the relative orientations of theCDR-like loops (CDR loop symmetry) for a variable domain be reproducedto a reasonable approximation in the relative orientation of thestructural loops. EP0640130B1 claims that such an approximation of theCDR-like loop symmetry does exist for the structural loops. However, itis doubtful that the relative orientation of the CDR-loops and thestructural loops is similar in sufficent detail and resolution;consequently it has not been described to date that it is actuallypossible to develop bispecific molecules by this technique.

EP0640130B1 exemplifies that R19.9 (a monoclonal murine antibodyspecific for the p-azobenzenearsonate) and 26-10 (monoclonal murineantibody specific for ouabain) were used as the framework providing theprimary CDR loops respectively, and the CDR-loops of murineanti-lysozyme antibody D1.3 were grafted into the structural loopregions. However, functional specificity after grafting is notdescribed.

Another example describes that the single chain antibody 26-10 specificfor ouabain could retain its ouabain-specificity after grafting two CDRsfrom the lysozyme-specific antibody into the structural loops of theouabain-specific single-chain Fv antibody fragment. However, it is notdescribed that the antibody fragment which was made according to thismethod had also lysozyme-binding specificity.

Grafting of Peptides into the Structural Loop Region

WO00244215A2 describes binding molecules comprising a specific targetbinding site and an Fc effector peptide. The Fc effector peptide is apeptide of up to 100 amino acids which interacts with effectormolecules. The effector peptide may, e.g. be inserted into the loopregions of an antibody provided that the ability to bind an antigen isnot adversely affected. The insertion of an effector peptide into anon-CDR loop of a CH1-domain of an immunoglobulin fragment isexemplified. The same insertion is not described for a variable domain.Every peptide grafted into a non-CDR loop according to this disclosurehas a high chance of being inactive due to the different structuralenvironment in which it has been placed. In addition, it may bedifficult to retain a specific CDR-loop conformation in a parentimmunoglobulin if a peptide is grafted into a structural loop of avariable domain. Consequently, it is not described that an effectorpeptide can be grafted into a variable domain without loss of eitherantigen binding or effector molecule binding.

PCT/EP2006/050059 describes a method of engineering an immunoglobulinwhich comprises a modification in a structural loop region to obtain anew antigen binding sites. This method is broadly applicable toimmunoglobulins and may be used to produce a series of immunoglobulinstargeting a variety of antigens.

US2005/266000A1 describes polypeptides comprising a variant heavy chainvariable framework domain (VFR). A VFR is part of the antigen bindingpocket or groove that may contact antigen. VFRs are part of the CDR loopregion, herein also called CDR region, and located at a variable domainat the side of the CDR loops to support the antigen binding via the CDRloop region. Framework loops other than VFR have not been mutated forthe purpose of engineering an antigen binding site.

It is an object of the present invention to provide immunoglobulins andvariable immunoglobulin domains with improved antigen binding propertiesand methods for engineering and manufacturing immunoglobulins with theimproved variable domains.

DESCRIPTION OF THE INVENTION

The object is met by the subject matter of the present invention.

The present invention relates to a method for engineering animmunoglobulin comprising a variable domain and at least onemodification in at least two structural loops of said immunoglobulin anddetermining the binding of said immunoglobulin to an epitope of anantigen, wherein the unmodified immunoglobulin does not significantlybind to said epitope, comprising the steps of:

-   -   providing a nucleic acid encoding an immunoglobulin comprising        at least two structural loops,    -   modifying at least one nucleotide residue of each of said        structural loops,    -   transferring said modified nucleic acid in an expression system,    -   expressing said modified immunoglobulin,    -   contacting the expressed modified immunoglobulin with an        epitope, and    -   determining whether said modified immunoglobulin binds to said        epitope.

The structural loops engineered according to the invention arepreferably located within one or two structural loop regions, in somecases the modifications are placed in even more than 2 structural loopregions.

In order to overcome the shortcomings of the state of the art asdescribed in WO00244215A2 and EP0640130B1 in engineering a binding siteinto a structural loop region of an antibody variable domain, thepresent invention provides methods to select specific binding of apolypeptide (i.e. a variable domain peptide comprising modifiedstructural loop regions) in its natural context (i.e. antibody variabledomain). In order to increase the number of potential structures ofvariant immunoglobulin domains which can be selected for antigen-bindingat least two structural loops or loop regions are modified according tothe invention. Thus it is possible to introduce and select for manydifferent modifications with minimal disturbance of the overall domainstructure. Another advantage of modifying at least two loops or loopregions is the enlarged surface area potentially able to interact with aspecific binding partner. The thus modified immunoglobulin variabledomains are selected for specific functions or binding. The inventionenables to engineer a modified immunoglobulin variable domain and amodified immunoglobulin binding with its modified structural loops orloop regions with high affinity binding and/or high specificity to abinding partner. The method allows selection of specific bindingmolecules with minimal modifications and without destruction of theoverall structure of the variable immunoglobulin domain.

To overcome the difficulty to retain a specific CDR-loop conformation ina parent immunoglobulin a structural loop of a variable domain ismodified according to the invention. It is thus the first time possibleto add to the antigen binding properties of an immunoglobulin withoutsignificant loss of antigen binding or even effector molecule binding ofa parent antibody. It is surprisingly possible to engineer theimmunoglobulin according to the invention without significantlyinterfering with the binding properties of a specific CDR-loopconformation, such as binding affinity, avidity and specificity, of theparent immunoglobulin.

A specific CDR-loop conformation provides for antigen-binding by CDRloops or a CDR loop region. Any immunoglobulin that is antigen bindingthrough such a CDR-loop conformation may be modified according to theinvention to obtain an additional binding site in a structural loop ofthe immunoglobulin, preferably while the specific CDR-loop conformationand the original antigen binding properties of the parent molecule maybe retained or unchanged. Preferably the binding properties of theCDR-loop conformation may be maintained to the extend that the bindingaffinity of the parent molecule is not significantly reduced, forinstance, the dissociation constant Kd is not significantly increased,meaning that the difference in. Kd is a factor preferably less than 10⁴,more preferably less than 10³, even more preferred less than 10², orless than 10.

In accordance with the present invention, one of the key features of thepresent invention is that the engineering of the immunoglobulin orimmunoglobulin variable domains takes place in regions which are notnormally involved in antigen binding, in other words, in regions otherthan the CDRs of an antibody variable domain. It was observed that thespecific fold of immunoglobulin domains allows the introduction ofrandom mutations in regions which are structurally analogous to the CDRsbut different in position in sequence and structure. The regionsidentified for modification according to the present invention are, likeCDRs, loop regions connecting the beta strands of the immunoglobulinfold. These structural loops or loop regions can be mutated as describedin the present invention without affecting the binding of the variabledomains of the immunoglobulin that is mediated through the CDR loops. Bymutating said structural loops or loop regions, a new molecular bindingsurface or binding pocket is generated that is similar in size and shapeto the binding surface or binding pocket formed by the CDR loops of thenatural antigen binding site of an antibody. Since the structural loopscan also be extended by the insertion of additional amino acids, thearchitecture of the newly generated binding site can be voluntarilyadjusted to the target to which it should bind. For example, deepbinding pockets which are especially suitable for the binding of smallmolecules can be preferentially formed by long loops, i.e. structuralloops with additional amino acids inserted in their sequence, whereasrather flat binding surfaces, which are well suited to bind to targetswith a large, flat molecular surface are preferentially formed when theresidues in the structural loops are mutated without the insertion ofadditional residues More specifically, it is described herein that byintroducing random mutations in the loops connecting beta strands A-B,C′-D and E-F of a human or humanized heavy chain variable domain,mutated domains can be selected that bind specifically to either humanserum albumin or to Fcgamma receptor III, which are not normallyrecognized and bound by human or humanized immunoglobulin variabledomains. The mutations introduced include mutations in which selectedamino acid residues in the wild-type sequence were replaced by randomlychosen residues, and they also include insertions of extra amino acidresidues in the loops mentioned above. Thus, preferably modifiedimmunoglobulins are provided according to the invention that areobtainable and produced according to the above described methods, andhave a binding site specific for an antigen, in particular specific fora serum albumin, cell receptors and complement factors, morespecifically human serum albumin and Fc receptors.

In particular, the present invention relates to a method for engineeringan immunoglobulin variable domain and an immunoglobulin containing sucha domain that is binding specifically to an epitope of an antigen.

Specifically the method according to the invention comprises the stepsof:

-   -   providing a nucleic acid encoding an immunoglobulin binding        specifically to at least one first epitope and is comprising at        least two structural loops or loop regions,    -   modifying at least one nucleotide residue of each of said        structural loops or loop regions encoded by said nucleic acid,    -   transferring said modified nucleic acid in an expression system,    -   expressing said modified immunoglobulin,    -   contacting the expressed modified immunoglobulin variable domain        with said at least one second epitope, and    -   determining whether said modified immunoglobulin variable domain        binds specifically to the second epitope.

This method preferably applies to immunoglobulin variable domainpeptides. More preferably the method according to this embodiment of theinvention relates to immunoglobulins that bind to said first epitopethrough their CDR-regions or specific CDR-loop conformation.

The method according to the invention refers further to at least onemodification in each of at least two structural loops or loop regions ofsaid immunoglobulin variable domain and determining the specific bindingof said structural loops or loop regions to at least one antigen,wherein the immunoglobulin variable domain containing an unmodifiedstructural loop or loop region does not specifically bind to such anantigen.

The term “immunoglobulin” as used herein is including immunoglobulins orparts, fragments or derivatives of immunoglobulins. Thus it includes an“immunoglobulin variable domain peptide” to be modified according to thepresent invention (as used herein the terms immunoglobulin and antibodyare interchangeable) as well as immunoglobulin variable domains or partsthereof that contain a structural loop, such as a minidomain, or astructural loop of such domains. The immunoglobulins can be used asisolated peptides or as combination molecules with other peptides. Insome cases it is preferable to use a defined modified structural loop ora structural loop region, or parts thereof, as isolated molecules forbinding or combination purposes. The “immunoglobulin variable domain” asdefined herein contains such immunoglobulin variable domain peptides orpolypeptides that may have specific binding characteristics uponmodifying and engineering. The peptides are homologous to immunoglobulinvariable domain sequences, and are preferably at least 5 amino acidslong, more preferably at least 10 or even at least 50 or 100 amino acidslong, and constitute at least partially the structural loop region orthe non-CDR loop region of the variable domain. Preferably the peptidesexclude those insertions that are considered non-functional amino acids,hybrid or chimeric CDR-regions or CDR-like regions and/or canonicalstructures of CDR regions. The binding characteristics relate tospecific epitope binding, affinity and avidity.

A derivative of an iummunoglobulin according to the invention is anycombination of one or more imunoglobulins of the invention and or afusion protein in which any domain or minidomain of the immunoglobulinof the invention may be fused at any position of one ore more otherproteins (such as other immunoglobulins, ligands, scaffold proteins,enzymes, toxins and the like). A derivative of the immunoglobulin of theinvention may also be obtained by recombination techniques or binding toother substances by various chemical techniques such as covalentcoupling, electrostatic interaction, disulphide bonding etc.

The other substances bound to the immunoglobulins may be lipids,carbohydrates, nucleic acids, organic and anorganic molecules or anycombination thereof (e.g. PEG, prodrugs or drugs). A derivative is alsoan immunoglobulin with the same amino acid sequence but made completelyor partly from non-natural or chemically modified amino acids.

The engineered molecules according to the present invention will beuseful as stand-alone proteins as well as fusion proteins orderivatives, most typically fused in such a way as to be part of largerantibody structures or complete antibody molecules, or parts thereofsuch as Fab fragments, Fc fragments, Fv fragments and others. It will bepossible to use the engineered proteins to produce molecules which arebispecific, trispecific, and maybe even carry more specificities at thesame time, and it will be possible at the same time to control andpreselect the valency of binding at the same time according to therequirements of the planned use of such molecules.

Another aspect of the present invention relates to an immunoglobulinwith at least one loop region besides the CDR-loop conformation of thevariable domain, characterised in that said at least one loop regioncomprises at least one amino acid modification forming at least onemodified loop region, wherein said at least one modified loop regionbinds specifically to at least one epitope of an antigen.

It is preferred to molecularly combine at least one modified antibodydomain, which is binding to the specific partner via the non-variablesequences or a structural loop, with at least one other bindingmolecule, which can be an antibody, antibody fragment, a solublereceptor, a ligand or another modified antibody domain.

The molecule that functions as a part of a binding pair that isspecifically recognized by the immunoglobulin according to the inventionis preferably selected from the group consisting of proteinaceousmolecules, nucleic, acids and carbohydrates.

The modified loops or loop regions of the immunoglobulins according tothe invention may specifically bind to any kind of binding molecules orstructures, in particular to antigens, proteinaceous molecules,proteins, peptides, polypeptides, nucleic acids, glycans, carbohydrates,lipids, small organic molecules, anorganic molecules, or combinations orfusions thereof. Of course, the modified immunoglobulins may comprise atleast two loops or loop regions whereby each of the loops or loopregions may specifically bind to different molecules or epitopes.

According to the present invention, binding regions to antigens orantigen binding sites of all kinds of cell surface antigens, may beintroduced into a structural loop of a given antibody structure.

Binding of the immunoglobulins according to the invention is preferablyon the one hand through CDR-regions, if the immunoglobulin contains sucha CDR-region, and on the other hand through an additional binding sitethat is formed by modifying at least two structural loops. Thosestructural loops are either placed on one or at least two variabledomains having one or more modified structural loops. Thus, animmunoglobulin according to the invention contains at least twomodifications in the structural loops of variable domains either throughat least one modification of at least two variable domains or at leasttwo modifications of at least one variable domain. Preferably themodified immunoglobulin thus exhibits a binding site either by changingthe primary structure of the protein or by changing the tertiarystructure to obtain a conformation specific binding site.

By analogy the immunoglobulin variable domains from any class ofimmunoglobulins and from immunoglobulins from any species are amenableto this type of engineering. Furthermore not only the specific loopstargeted in the examples of the present invention can be manipulated,but any loop connecting beta strands in immunoglobulin variable domainscan be manipulated in the same way.

Engineered immunoglobulins or, immunoglobulin variable domains from anyorganism and from any class of immunoglobulin can be used according tothe present invention either as such (as single domains), or as part ofa larger molecule. For example, they can be part of an intactimmunoglobulin, which accordingly would have its “normal” antigenbinding region formed by at least one of the 6 CDRs and the new,engineered antigen binding site. Like this, a multi-specific, e.g.bispecific, or trispecific immunoglobulin could be generated. Theengineered immunoglobulin domains can also be part of any fusionprotein.

Immunoglobulins or immunoglobulin variable domains according to theinvention may be complete antibody molecules or part of antibodies, suchas IgG, IgA, IgM, IgD, IgE and the like. The immunoglobulins orimmunoglobulin variable domains of the invention may also contain orconsist of a functional antibody fragment such as Fab, Fab2, scFv, Fv,or parts thereof, or other derivatives or combinations of theimmunoglobulins such as minibodies, domains of the heavy and lightchains of the variable region (such as Fd, VL, including Vlambda andVkappa, VH, VHH) as well as mini-domains consisting of two beta-strandsof an immunoglobulin domain connected by at least two structural loops(see for example Laffly et al (2005) Hum Antibodies. 2005;14:33-55), asisolated domains or in the context of naturally associated molecules.

The modified immunoglobulin according to the invention is possiblyfurther combined with one or more modified immunoglobulins or withunmodified immunoglobulins, or parts thereof, to obtain a combinationimmunoglobulin. Combinations are preferably obtained by recombinationtechniques, but also by association through adsorption, electrostaticinteractions or the like, or else through chemical binding with orwithout a linker. The preferred linker sequence is either a naturallinker sequence or a functionally suitable artificial sequence.

It is understood that the term “immunoglobulin”, “immunoglobulinvariable domain peptide” and “immunoglobulin variable domain” includesderivatives as well. A derivative is any part or combination of one ormore immunoglobulins and/or a fusion protein in which any domain orminidomain of the immunoglobulin as obtained according to the inventionmay be combined or fused at any position with one ore more otherpeptides or proteins (including but not limited to otherimmunoglobulins, immunoglobulin domains, Fc parts, ligands, scaffoldproteins, enzymes, toxins, serum proteins and the like). A derivative ofthe immunoglobulin of the invention may also be obtained by binding theunmodified or modified immunoglobulin or immunoglobulin variable domainof the invention to other substances by various chemical techniques suchas covalent coupling, electrostatic interaction, disulphide bonding etc.

The other substances bound to the immunoglobulin or immunoglobulinvariable domain may be lipids, carbohydrates, nucleic acids, organic andanorganic molecules or any combination thereof (e.g. PEG, pro-drugs ordrugs). A derivative is also an immunoglobulin or immunoglobulinvariable domain with the same amino acid sequence but made completely orpartly from non-natural, artificial or chemically modified amino acids.

The engineered molecules according to the present invention will beuseful as stand-alone proteins as well as fusion proteins orderivatives, most typically fused before or after modification in such away as to be part of larger antibody structures or complete antibodymolecules, or parts thereof. Immunoglobulins according to the inventionmay thus also consist of or comprise Fab fragments, Fc fragments, Fvfragments, single chain antibodies, in particular single-chain Fvfragments, bi- or multispecific scFv, diabodies, multibodies,multivalent or multimers of immunoglobulin domains and others. It willbe possible to use the engineered proteins to produce molecules whichare monospecific, bispecific, trispecific, and molecules that may evencarry more specificities. By the invention it is possible to control andpreselect the valency of binding at the same time according to therequirements of the planned use of such molecules.

According to the present invention, one or more binding sites toantigens or antigen binding sites to one or more antigens may beintroduced into a structural loop or loop region of a given antibodyvariable domain structure. The antigens may be naturally occurringmolecules or chemically synthesized molecules or recombinant molecules,either in solution or in suspension, e.g. located on or in particlessuch as solid phases, on or in cells or on viral surfaces. It wassurprising that the binding of an immunoglobulin to an antigen could beachieved according to the invention, even when the antigen is stilladhered or bound to molecules and structures that would hinder itsbinding. By employing the target antigen in its selected or naturalcontext and structure for selecting the modified and designedimmunoglobulin, it is possible to identify and obtain those modifiedimmunoglobulins that are best suitable for the purpose of diagnostic ortherapeutic use.

The term “antigen” as used herein shall comprise molecules selected fromthe group consisting of allergens, tumor associated antigens, selfantigens including albumin, T cell receptors, FcRn, cell surfacereceptors, enzymes, Fc-receptors, proteins of the complement system,serum molecules, bacterial antigens, fungal antigens, protozoan antigenand viral antigens, also molecules responsible for transmissiblespongiform encephalitis (TSE), such as prions, infective or not, andmarkers or molecules that relate to Alzheimer disease. Antigens can betargeted and bound by the immunoglobulins or immunoglobulin variabledomains as engineered according to the invention through at least partof a modified structural loop.

In a preferred embodiment the immunoglobulin is binding with itsmodified structural loops specifically to at least two such epitopesthat are identical or differ from each other, either of the same antigenor of different antigens.

For example, the method according to the invention refers to engineeringan immunoglobulin or immunoglobulin variable domain that is bindingspecifically to at least one first epitope and comprising at least onemodification in each of at least two structural loops or loop regions ofsaid immunoglobulin, and determining the specific binding of said loopsor loop regions to at least one second epitope, the epitope beingselected from the group of antigens as mentioned above, wherein theunmodified structural loop or loop region (non-CDR region) does notspecifically bind to said at least one second epitope.

The term antigen as used according to the present invention shall inparticular include all antigens and target molecules capable of beingrecognised by a binding site of an immunoglobulin or an antibodystructure, as a whole target molecule or as a fragment of such molecule(especially substructures of targets, generally referred to as“epitopes”).

Preferred antigens as targeted by the immunoglobulins according to theinvention are those antigens or molecules, which have already beenproven to be or are capable of being immunogenic, bound by immuneresponse factors, or else immunologically or therapeutically relevant,especially those, for which a clinical efficacy has been tested.

The term “antigen” according to the present invention shall meanmolecules or structures known to interact or capable of interacting withthe CDR-loop region of immunoglobulins. Structural loop regions of theprior art referring to native antibodies, do not interact with antigensbut rather contribute to the overall structure and/or to the binding toeffector molecules. Only upon engineering according to the inventionstructural loops may form antigen binding pockets without involvement ofCDR loops or the CDR region.

According to a preferred embodiment the antigen bound by theimmunoglobulin according to the invention is a cell surface antigen. Theterm “cell surface antigens” shall include all antigens or capable ofbeing recognised by an antibody structure on the surface of a cell, andfragments of such molecules. Preferred “cell surface antigens” are thoseantigens, which have already been proven to be or which are capable ofbeing immunologically or therapeutically relevant, especially those, forwhich a preclinical or clinical efficacy has been tested. Those cellsurface molecules are specifically relevant for the purpose of thepresent invention, which mediate cell killing activity. Upon binding ofthe immunoglobulin according to the invention to at least two of thosecell surface molecules the immune system provides for cytolysis or celldeath, thus a potent means for attacking human cells may be provided.

Preferably the antigen is selected from cell surface antigens, includingreceptors, in particular from the group consisting of erbB receptortyrosine kinases (such as EGFR, HER2, HER3 and HER4, but not limited tothese), molecules of the TNF-receptor superfamily, such as Apo-1receptor, TNFR1, TNFR2, nerve growth factor receptor NGFR, CD40, T-cellsurface molecules, T-cell receptors, T-cell antigen OX40, TACI-receptor,BCMA, Apo-3, DR4, DR5, DR6, decoy receptors, such as DcR1, DcR2, CAR1,HVEM, GITR, ZTNFR-5, NTR-1, TNFL1 but not limited to these molecules,B-cell surface antigens, such as CD10, CD19, CD20, CD21, CD22, antigensor markers of solid tumors or hematologic cancer cells, cells oflymphoma or leukaemia, other blood cells including blood platelets, butnot limited to these molecules.

According to a further preferred embodiment of the present invention theantigen or the molecule binding to the modified structural loop or loopregion is selected from the group consisting of tumor associatedantigens, in particular EpCAM, tumor-associated glycoprotein-72(TAG-72), tumor-associated antigen CA 125, Prostate specific membraneantigen (PSMA), High molecular weight melanoma-associated antigen(HMW-MAA), tumor-associated antigen expressing Lewis Y relatedcarbohydrate, Carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM, mucinMUC1, MUC18 and cytokeratin tumor-associated antigen, bacterialantigens, viral antigens, allergens, allergy related molecules IgE, cKITand Fc-epsilon-receptorI, IRp60, IL-5 receptor, CCR3, red blood cellreceptor (CR1), human serum albumin, mouse serum albumin, rat serumalbumin, neonatal Fc-gamma-receptor FcRn, Fc-gamma-receptors Fc-gammaRI, Fc-gamma-RII, Fc-gamma RIII, Fc-alpha-receptors,Fc-epsilon-receptors, fluorescein, lysozyme, toll-like receptor 9,erythropoietin, CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18,CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32, CD33 (p67protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3,IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15,IL-18, IL-23, interferon alpha, interferon beta, interferon gamma;TNF-alpha, TNFbeta2, TNFalpha, TNFalphabeta, TNF-R1, TNF-RII, FasL,CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1,OX40L, TRAIL Receptor-1, A1 Adenosine Receptor, Lymphotoxin BetaReceptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, integrin beta1,integrin beta2, integrin alpha4/beta7, integrin alpha2, integrin alpha3,integrin alpha4, integrin alpha5, integrin alpha6, integrin alphav,alphaVbeta3 integrin, FGFR-3, Keratinocyte Growth Factor, VLA-1, VLA-4,L-selectin, anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor,B7-1, B7-2, VNRintegrin, TGFbeta1, TGFbeta2, eotaxin1, BLyS(B-lymphocyte Stimulator), complement C5, IgE, IgA, IgD, IgM, IgG,factor VII, CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3(ErbB-3), Her4 (ErbB4), Tissue Factor, VEGF, VEGFR, endothelin receptor,VLA-4, carbohydrates such as blood group antigens and relatedcarbohydrates, Galili-Glycosylation, Gastrin, Gastrin receptors, tumorassociated carbohydrates, Hapten NP-cap or NIP-cap, T cell receptoralpha/beta, E-selectin, P-glycoprotein, MRP3, MRP5,glutathione-S-transferase pi (multi drug resistance proteins),alpha-granule membrane protein (GMP) 140, digoxin, placental alkalinephosphatase (PLAP) and testicular PLAP-like alkaline phosphatase,transferrin receptor, Heparanase I, human cardiac myosin, GlycoproteinIIb/IIIa (GPIIb/IIIa), human cytomegalovirus (HCMV) gH envelopeglycoprotein, HIV gp120, HCMV, respiratory syncital virus RSV F, RSVFFgp, VNRintegrin, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120 V3 loop,respiratory syncytial virus (RSV) Fgp, Herpes simplex virus (HSV) gDglycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein,Clostridium perfringens toxin and fragments thereof.

Preferably, the antigen is selected from the group consisting ofpathogen antigen, tumor associated antigen, enzyme, substrate, selfantigen, organic molecule or allergen. More preferred antigens areselected from the group consisting of viral antigens, bacterial antigensor antigens from pathogens of eukaryots or phages. Preferred viralantigens include HAV-, HBV-, HCV-, HIV I-, HIV II-, Parvovirus-,Influenza-, HSV-, Hepatitis Viruses, Flaviviruses, Westnile Virus, EbolaVirus, Pox-Virus, Smallpox Virus, Measles Virus, Herpes Virus,Adenovirus, Papilloma Virus, Polyoma Virus, Parvovirus, Rhinovirus,Coxsackie virus, Polio Virus, Echovirus, Japanese Encephalitis virus,Dengue Virus, Tick Borne Encephalitis Virus, Yellow Fever Virus,Coronavirus, respiratory syncytial virus, parainfluenza virus, La CrosseVirus, Lassa Virus, Rabies Viruse, Rotavirus antigens; preferredbacterial antigens include Pseudomonas-, Mycobacterium-,Staphylococcus-, Salmonella-, Meningococcal-, Borellia-, Listeria,Neisseria-, Clostridium-, Escherichia-, Legionella-, Bacillus-,Lactobacillus-, Streptococcus-, Enterococcus-, Corynebacterium-,Nocardia-, Rhodococcus-, Moraxella-, Brucella, Campylobacter-,Cardiobacterium-, Francisella-, Helicobacter-, Haemophilus-,Klebsiella-, Shigella-, Yersinia-, Vibrio-, Chlamydia-, Leptospira-,Rickettsia-, Mycobacterium-, Treponema-, Bartonella-antigens. Preferredeukaryotic antigens of pathogenic eukaryotes include antigens fromGiardia, Toxoplasma, Cyclospora, Cryptosporidium, Trichinella, Yeasts,Candida, Aspergillus, Cryptococcus, Blastomyces, Histoplasma,Coccidioides.

The modified immunoglobulin according to the present invention maypreferably bind to one of the molecules disclosed above. These moleculescomprise also allergens and haptens.

Substructures of antigens are generally referred to as “epitopes” (e.g.B-cell epitopes, T-cell epitopes), as long as they are immunologicallyrelevant, i.e. are also recognisable by natural or monoclonalantibodies. The term “epitope” according to the present invention shallmean a molecular structure which may completely make up a specificbinding partner or be part of a specific binding partner to the bindingdomain or the immunoglobulin of the present invention.

Chemically, an epitope may either be composed of a carbohydrate, apeptide, a fatty acid, an anorganic substance or derivatives thereof andany combinations thereof. If an epitope is a peptide or polypeptide,there will usually be at least 3 amino acids, preferably 8 to 50 aminoacids, and more preferably between about 10-20 amino acids included inthe peptide. There is no critical upper limit to the length of thepeptide, which could comprise nearly the full length of the polypeptidesequence. Epitopes can either be linear or conformational epitopes. Alinear epitope is com-prised of a single segment of a primary sequenceof a polypeptide chain. Linear epitopes can be contiguous oroverlapping. Conformational epitopes are comprised of amino acidsbrought together by folding of the polypeptide to form a tertiarystructure and the amino acids are not necessarily adjacent to oneanother in the linear sequence.

Specifically, epitopes are at least part of diagnostically relevantmolecules, i.e. the absence or presence of an epitope in a sample isqualitatively or quantitatively correlated to either a disease or to thehealth status or to a process status in manufacturing or toenvironmental and food status. Epitopes may also be at least part oftherapeutically relevant molecules, i.e. molecules which can be targetedby the specific binding domain which changes the course of the disease.

Besides the antigen binding sites of the immunoglobulin as engineeredaccording to the invention further binding capacities may be introducedaside from or into the structural loop regions of variable domains, e.g.binding capacities for other antigens, small molecules, for drugs orenzymes, catalytic sites of enzymes or enzyme substrates or the bindingto a transition state analogue of an enzyme substrate.

Preferably the new antigen binding site in the structural loops isforeign to the unmodified immunoglobulin variable domain. Thus foreigntargets like effector molecules, serum proteins or Fc-receptors or cellsurface receptors, which are not bound by variable domains by nature,are preferred as antigens or binding molecules bound by theimmunoglobulin variable domains according to the invention.

In this contect the term “foreign” means that the antigen is notrecognized by the specific CDR binding region or other natural orintrinsic binding regions of the immunoglobulin. A foreign bindingpartner, but not the natural binding partner of an immunoglobulin, maythus be bound by the newly formed antigen binding site of a structuralloop. This means that a natural binding partner, such as an Fc-receptoror an effector of the immune system, is not considered to be bound bythe antigen binding site foreign to the unmodified immunoglobulin.

As used herein, the term “specifically binds” or “specific binding”refers to a binding reaction, which is determinative of the cognateligand of interest in a heterogeneous population of molecules. Thus,under designated conditions (e.g. immunoassay conditions), the specifiedantibody variable domain binds to its particular “target” and does notbind in a significant amount to other molecules present in a sample. Thespecific binding means that binding is selective in terms of targetidentity, high, medium or low binding affinity or avidity, as selected.Selective binding is usually achieved if the binding constant or bindingdynamics is at least 10 fold different.

The term “expression system” refers to nucleic acid molecules containinga desired coding sequence and control sequences in operable linkage, sothat hosts transformed or transfected with these sequences are capableof producing the encoded proteins. In order to effect transformation,the expression system may be included on a vector; however, the relevantDNA may then also be integrated into the host chromosome. Alternatively,an expression system can be used for in vitro transcription/translation.

A “structural loop” or “non-CDR-loop” according to the present inventionis to be understood in the following manner: immunoglobulins are made ofdomains with a so called immunoglobulin fold. In essence, anti-parallelbeta sheets are connected by loops to form a compressed antiparallelbeta barrel. In the variable region, some of the loops of the domainscontribute essentially to the specificity of the antibody, i.e. thebinding to an antigen. These loops are called CDR-loops.

The CDR loops are located within the CDR loop region, which may in somecases also the variable framework region (called “VFR”) adjacent to theCDR loops. It is known that VFRs may contribute to the antigen bindingpocket of an antibody, which generally is mainly determined by the CDRloops. Thus, those VFRs are considered as part of the CDR loop region,and would not be appropriately used for the purpose of the invention.Contrary to those VFRs within the CDR loop region or located proximal tothe CDR loops, other VFRs of variable domains would be particularlysuitable to be used according to the invention. Those are the structuralloops of the VFRs located opposite to the CDR loop region, or at theC-terminal side of a variable immunoglobulin domain.

All loops of antibody variable domains outside the CDR loop region arerather contributing to the structure of the molecule. These loops aredefined herein as structural loops or non-CDR-loops.

All numbering of the amino acid sequences of the, immunoglobulins isaccording to the IMGT numbering scheme (IMGT, the internationalImMunoGeneTics information system@imgt.cines.fr; http://imgt.cines.fr;Lefranc et al., 1999, Nucleic Acids Res. 27: 209-212; Ruiz et al., 2000Nucleic Acids Res. 28: 219-221; Lefranc et al., 2001, Nucleic Acids Res.29: 207-209; Lefranc et al., 2003, Nucleic Acids Res. 31: 307-310;Lefranc et al., 2005, Dev Comp Immunol 29:185-203).

According to a preferred embodiment of the present invention theimmunoglobulin variable domain is of human, camelid, rabbit; chicken,rat, dog, horse, sheep or murine origin.

Since the modified immunoglobulin may be employed for various purposes,in particular in pharmaceutical compositions, the immunoglobulin ispreferably of human, camelid or murine origin.

Of course, the modified immunoglobulin may also be a humanized or achimeric immunoglobulin. In the most preferred embodiment of theinvention the modified variable domain is of human origin or a humanizedversion of a variable domain of any species.

A humanized immunoglobulin variable domain has at least about 50% aminoacid sequence identity, preferably at least about 55% amino acidsequence identity, more preferably at least about 60% amino acidsequence identity, more preferably at least about 65% amino acidsequence identity, more preferably at least about 70% amino acidsequence identity, more preferably at least about 75% amino acidsequence identity, more preferably at least about 80% amino acidsequence identity, more preferably at least about 85% amino acidsequence identity, more preferably at least about 90% amino acidsequence identity, more preferably at least about 95% amino acidsequence identity to a native human immunoglobulin variable domainsequence.

A humanized immunoglobulin variable domain has furthermore at leastabout 50% amino acid sequence identity, preferably at least about 55%amino acid sequence identity, more preferably at least about 60% aminoacid sequence identity, more preferably at least about 65% amino acidsequence identity, more preferably at least about 70% amino acidsequence identity, more preferably at least about 75% amino acidsequence identity, more preferably at least about 80% amino acidsequence identity, more preferably at least about 85% amino acidsequence identity, more preferably at least about 90% amino acidsequence identity, more preferably at least about 95% amino acidsequence identity when comparing all surface accessible amino acids tothe surface accessible amino acids of a native human immunoglobulinvariable domain sequence.

The preferred homology or sequence identities specifically relates tothose sequences of the framework region.

The preferred immunoglobulin according to the invention comprises adomain that has at least 50% homology with the unmodified domain.

The term “homology” indicates that polypeptides have the same orconserved residues at a corresponding position in their primary,secondary or tertiary structure. The term also extends to two or morenucleotide sequences encoding the homologous polypeptides.

“Homologous immunoglobulin domain” means an immunoglobulin domainaccording to the invention having at least about 50% amino acid sequenceidentity with regard to a full-length native sequence immunoglobulindomain sequence or any other fragment of a full-length immunoglobulindomain sequence as disclosed herein. Preferably, a homologousimmunoglobulin domain will have at least about 50% amino acid sequenceidentity, preferably at least about 55% amino acid sequence identity,more preferably at least about 60% amino acid sequence identity, morepreferably at least about 65% amino acid sequence identity, morepreferably at least about 70% amino acid sequence identity, morepreferably at least about 75% amino acid sequence identity, morepreferably at least about 80% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 95% amino acid sequence identity to a nativeimmunoglobulin domain sequence, or any other specifically definedfragment of a full-length immunoglobulin domain sequence as disclosedherein.

“Percent (%) amino acid sequence identity” with respect to theimmunoglobulin domain sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the specific immunoglobulinvariable domain sequence, after aligning the sequence and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared.

% amino acid sequence identity values may be obtained as described belowby using the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=l, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of theimmunoglobulin variable domain of interest having a sequence derivedfrom the native immunoglobulin variable domain and the comparison aminoacid sequence of interest (i.e., the sequence against which, theimmunoglobulin variable domain of interest is being compared which maybe the unmodified immunoglobulin variable domain) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of thenon-randomized parts of the immunoglobulin variable domain of interest.For example, in the statement “a polypeptide comprising an amino acidsequence X which has or having at least 80% amino acid sequence identityto the amino acid sequence Y”, the amino acid sequence A is thecomparison amino acid sequence of interest and the amino acid sequence Bis the amino acid sequence of the immunoglobulin variable domain ofinterest.

In a preferred embodiment the polypeptide according to the invention isa bispecific antibody or a bispecific single chain antibody or abispecific Fab or a bispecific sdAb. Further preferred is that thepolypeptide comprises a bispecific domain or a part thereof. Specificexamples refer to Fab or dAb molecules with additional functionalitythrough the new binding site at the opposite side of the CDR region. Thepreferred immunoglobulin according to the invention, which is a Fabmolecule, may, for example, contain one or two additional binding sitesat the C-terminal loop side of a CH1 and/or a CL domain. The preferredimmunoglobulin according to the invention, which is a dAb molecule, may,for example, contain an additional binding site at the C-terminal loopside of a Vh, Vhh or Vl domain. By such additional binding sitesadditional functionalities, like prolonged half life (e.g. throughbinding to an FcRn or serum proteins, such as albumin or IgG), oreffector function (e.g. through binding to T cell receptors, C1q orCD64) can be added to the molecules. According to such examples thededicated Fab or dAb libraries, would be prepared with an appropriatesize to enable selection of the specific binders to the specific bindingpartners.

The preferred variable domain according to the invention is selectedfrom the group of VH, VL, including Vkappa and Vlambda, VHH andcombinations thereof. It turned out that those modifications are ofspecific advantage when brought into the loops or loop regions of a VH,a Vkappa, a Vlambda or a VHH, and the modified loops or loop regionscomprise at least one modification within amino acids 7 to 21, aminoacids 25 to 39, amino acids 41 to 81, amino acids 83 to 85, amino acids89 to 103 or amino acids 106 to 117.

The structural loops or loop regions of the immunoglobulin or variabledomain of the immunoglobulin of human or humanized origin as modifiedaccording to the invention are selected preferably from the structuralloops that comprise amino acids 8 to 20, amino acids 44 to 50, aminoacids 67 to 76 and amino acids 89 to 101, most preferably amino acidpositions 12 to 17, amino acid positions 45 to 50, amino acid positions69 to 75 and amino acid positions 93 to 98.

In another preferred embodiment a modification in the structural loopsor loop regions comprising amino acids 93 to 98 is combined with amodification in the structural loops or loop regions comprising aminoacids 8 to 20.

The above identified amino acid regions of the respectiveimmunoglobulins are loops or loop regions specified to be suitable formodification purposes according to the invention. Preferablycombinations of such modifications, e.g. in at least two of thespecified loops or loop regions, are engineered into the immunoglobulinaccording to the invention.

Preferably, a modification in the structural loop or loop regioncomprising amino acids 93 to 98 is combined with a modification in oneor more of the other structural loops.

In a preferred embodiment a modification in the structural loop or loopregion comprising amino acids 93 to 98 is combined with a modificationin the structural loop region comprising amino acids 69 to 75.

Most preferably each of the structural loops comprising amino acids 93to 98, amino acids 69 to 75 and amino acids 8 to 20 contain at least oneamino acid modification.

In another preferred embodiment each of the structural loops comprisingamino acids 93 to 98, amino acids 69 to 75, amino acids 44 to 50 andamino acids 8 to 20 contain at least one amino acid modification.

According to a preferred embodiment of the present invention thestructural loops or loop regions of an immunoglobulin or the variabledomain of the immunoglobulin of murine origin, e.g. a VH, comprise aminoacids 6 to 20, amino acids 44 to 52, amino acids 67 to 76 and aminoacids 92 to 101.

According to another preferred embodiment of the present invention thestructural loops or loop regions of an immunoglobulins or the variabledomain of the immunoglobulin of camelid origin, e.g. VHH, comprise aminoacids 7 to 18, amino acids 43 to 55, amino acids 68 to 75 and aminoacids 91 to 101.

The variable domains of camelid origin or humanized variants of camelidorigin, have the advantage that they could easily be combined with othervariable domains, for instance with other VHH of camelid origin,modified or native. The possible combination of VHH of camelid origin isthe basis for multivalent immunoglobulins. Thus, according to theinvention specific modified variable domains of camelid origin aremultivalent combinations, preferably with at least 3, more preferablywith at least 4 or 5 valencies or VHHs.

Preferably, the new antigen binding sites in the structural loops areintroduced into the immunoglobulin encoded by the selected nucleic acidby substitution, deletion and/or insertion of at least one nucleotide.

According to another preferred embodiment of the present invention themodification of at least one nucleotide in each of at least twostructural loops or loop regions result in a substitution, deletionand/or insertion in the immunoglobulin or immunoglobulin variable domainencoded by said nucleic acid.

The modification of the at least two loops or loop regions of animmunoglobulin or antibody variable domain may result in a substitution,deletion and/or insertion of 2 or more amino acids, preferably pointmutations, change of amino acids of whole loops, more preferred thechange of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, up to 30amino acids. However, the maximum number of amino acids inserted into astructural loops or loop regions of an immunoglobulin or immunoglobulinvariable domain may in specific cases not exceed the number of 30,preferably 25, more preferably 20, amino acids.

Thereby the modified sequence comprises amino acids not included in theconserved regions of the structural loops, the newly introduced aminoacids being naturally occurring, but foreign to the site ofmodification, or substitutes of naturally occurring amino acids. Whenthe foreign amino acid is selected from a specific group of amino acids,such as amino acids with specific polarity, or hydrophobicity, a libraryenriched in the specific group of amino acids at the randomizedpositions can be obtained according to the invention. Such libraries arealso called “focused” libraries.

The at least two loops or loop regions are preferably mutated ormodified by random, semi-random or, in particular, by site-directedrandom mutagenesis methods.

A preferred method to introduce modifications is site directed randommutation. With this method two or more specific amino acid residues ofthe loops are exchanged or introduced using randomly generated insertsinto such structural loops. Alternatively preferred is the use ofcombinatorial approaches.

In another preferred embodiment at least three structural loops or loopregions of an immunoglobulin or immunoglobulin variable domain aremutated or modified by random, semi-random or, in particular, bysite-directed random mutagenesis methods.

These methods may be used to make amino acid modifications at desiredpositions of the immunoglobulins or immunoglobulin variable domain ofthe present invention. In these cases positions are chosen randomly, oramino acid changes are made using certain rules. For example certainresidues may be mutated to any amino acids, whereas other residues maybe mutated to a restricted set of amino acids. This can be achieved in astepwise fashion by alternating of cycles of mutation and selection orsimultaneously.

The randomly modified nucleic acid molecule may comprise the hereinidentified repeating units, which code for all known naturally occurringamino acids or a subset thereof. Those libraries that contain modifiedsequences wherein a specific subset of amino acids are used formodification purposes are called “focused” libraries. The member of suchlibraries have an increased probability of an amino acid of such asubset at the modified position, which is at least two times higher thanusual, preferably at least 3 times or even at least 4 times higher. Suchlibraries have also a limited or lower number of library members, sothat the number of actual library members reaches the number oftheoretical library members. In some cases the number of library membersof a focused library is not less than 10³ times the theoretical number,preferably not less than 10² times, most preferably not less than 10times.

A library according to the invention may be designed as a librarycontaining or consisting of library members of a specific immunoglobulinformat. Those libraries that are consisting of specific immunoglkobulinmolecular format are called dedicated library for the purpose of thisinvention. Dedicated libraries preferably contain a majority of thespecific formats, at least 50%, preferably at least 60%, more preferredat least 70%, more preferred at least 80%, more preferred at least 90%,or those that essentially consist of specific antibody formats. Specificantibody formats are preferred, such that the preferred libraryaccording to the invention it is selected from the group consisting of aVH library, VHH library, Vkappa library, Vlambda library, Fab library, aCH1/CL library and a CH3 library. Libraries characterized by the contentof composite molecules containing more than one antibody domains, suchas an IgG library or Fc library are specially preferred. Other preferredlibraries are those containing T-cell receptors, forming T-cell receptorlibraries. Further preferred libraries are epitope libraries, whereinthe fusion protein comprises a molecule with a variant of an epitope,also enabling the selection of competitive molecules having similarbinding function, but different functionality. Exemplary is a TNFalphalibrary, wherein trimers of the TNFalpha fusion protein are displayed bya single genetic package.

However, the maximum number of amino acids inserted into a loop or loopregion of an immunoglobulin preferably may not exceed the number of 30,preferably 25, more preferably 20 amino acids at a maximum. Thesubstitution and the insertion of the amino acids occurs preferablyrandomly or semi-randomly using all possible amino acids or a selectionof preferred amino acids for randomization purposes, by methods known inthe art and as disclosed in the present patent application.

The site of modification may be at a specific single structural loop ora structural loop region. A loop region usually is composed of at leasttwo, preferably at least 3 or at least 4 loops that are adjacent to eachother, and which may contribute to the binding of an antigen throughforming an antigen binding site or antigen binding pocket. It ispreferred that the one or more sites of modification are located withinthe area of 10 amino acids, more preferably within 20, 30, 40, 50, 60,70, 80, 90 up to 100 amino acids, in particular within a structural loopregion to form a surface or pocket where the antigen can stericallyaccess the loop regions.

The at least one loop or loop region is preferably mutated or modifiedto produce libraries, preferably by random, semi-random or, inparticular, by site-directed random mutagenesis methods, in particularto delete, exchange or introduce randomly generated inserts intostructural loops. Alternatively preferred is the use of combinatorialapproaches. Any of the known mutagenesis methods may be employed, amongthem cassette mutagenesis. These methods may be used to make amino acidmodifications at desired positions of the immunoglobulin of the presentinvention. In some cases positions are chosen randomly, e.g. with eitherany of the possible amino acids or a selection of preferred amino acidsto randomize loop sequences, or amino acid changes are made usingsimplistic rules. For example all residues may be mutated preferably tospecific amino acids, such as alanine, referred to as amino acid oralanine scanning. Such methods may be coupled with more sophisticatedengineering approaches that employ selection methods to screen higherlevels of sequence diversity.

A preferred method according to the invention refers to a randomlymodified nucleic acid molecule coding for an immunoglobulin,immunoglobulin domain or a part thereof which comprises at least onenucleotide repeating unit within a structural loop coding region havingthe sequence 5′-NNS-3′, 5′-NNN-3′, 5′-NNB-3′ or 5′-NNK-3′. In someembodiments the modified nucleic acid comprises nucleotide codonsselected from the group of TMT, WMT, BMT, RMC, RMG, MRT, SRC, KMT, RST,YMT, MKC, RSA, RRC, NNK, NNN, NNS or any combination thereof (the codingis according to IUPAC).

The randomly modified nucleic acid molecule may comprise the aboveidentified repeating units, which code for all known naturally occurringamino acids or a subset thereof.

The modification of the nucleic acid molecule may be performed byintroducing synthetic oligonucleotides into a larger segment of nucleicacid or by de novo synthesis of a complete nucleic acid molecule.Synthesis of nucleic acid may be performed with tri-nucleotide buildingblocks which would reduce the number of nonsense sequence combinationsif a subset of amino acids is to be encoded (e.g. Yanez et al. NucleicAcids Res. (2004) 32:e158; Virnekas et al. Nucleic Acids Res. (1994)22:5600-5607).

Preferably the positions to be modified are surface exposed amino acids.Surface exposition of amino acids of structural loops can be judged fromknown protein structures of antibody variable domains and by analogy orhomology for such amino acid sequences for which no experimentallydetermined structure is available.

In a preferred embodiment of the invention the modifications introducedinto the at least two structural loops comprise at least 1, 2, 3, 4, 5or 6 foreign amino acids or amino acids not naturally occurring at therespective site of the structural loop of the non-modifiedimmunoglobulin or immunoglobulin variable domain.

The modification of amino acids may preferentially be biased in order tointroduce into structural loops or loop regions amino acids which areknown to be frequently involved in protein-protein interactions (e.g.Lea & Stewart (1995) FASEB J. 9:87-93; Fellhouse et al. (2006) J. Mol.Biol. 357:100-114; Adib-Conquuy et al. (1998) International Immunology10:341-346; Lo Conte et al. (1999) J. Mol. Biol. 285:2177-2198; Zemlinet al. (2003) J. Mol. Biol. 334:733-749).

According to one embodiment of the invention an immunoglobulinobtainable by the method according to the invention is used for thepreparation of a library with library members displaying or encoding theimmunoglobulins according to the invention, specifically a library ofproteins, fusion proteins, cells, in particular microbial cells, likebacterial or yeast cells, phages, viruses, nucleic acids or ribosomes.

The following specification not only refers to libraries of polypeptideor protein variants, but, of course, also to the alternative librariesused for expressing the immunoglobulins according to the invention, e.g.as mentioned above.

In one preferred embodiment, a library of polypeptide variantscomprising immunoglobulins or immunoglobulin variable domains of theinvention is used as a pool for selection wherein the modificationscontain or introduce at least one, more preferably at least two aminoacids per modified structural loop out of the group of amino acidstryptophane, tyrosine, phenylalanine, histidine, isoleucine, serine,methionine, alanine and asparagine.

It turned out that according to the invention a variant variable domainpolypeptide can be provided with specific mutations that are foreign tothe native polypeptides. Any of the amino acids tryptophane, tyrosine,phenylalanine, histidine, isoleucine, serine, methionine, alanine andasparagines are not present in the structural loops of human nativeimmunoglobulins, thus are considered as “foreign”. A variant polypeptideaccording to the invention may contain at least two of said foreignamino acids in the structural loops, by modification of at least onestructural loop and to make up a binding site.

If the modified immunoglobulin or immunoglobulin variable domain is ofhuman origin or a humanized immunoglobulin,variable domain, preferredmodifications are the incorporation of least one tyrosine in any one ofthe positions 12 to 17, 45 to 50, 69 to 75 and 93 to 98, and/or at leastone tryptophane in any one of the positions 12 to 17, 45 to 50, 69, 71to 75, 93 to 94 and 96 to 98, and/or at least one histidine in any oneof the positions 12 to 17, 46, 47, 49, 50, 69 to 74 and 93 to 98, and/orat least one asparagine in any one of the positions 12 to 17, 45 to 47,49, 50, 70 to 73, 75, 94 to 96 and 98, and/or at least one methionine inany one of the positions 12 to 17, 46 to 50, 69 to 71, 73 to 75, 93, 95,96 and 98, and/or at least one serine in any one of the positions 13,71, 75, 94, 95 and 98, and/or at least one isoleucine in any one of thepositions 12, 14 to 17, 45 to 50, 69, 70, 72 to 75, 93 and 96 to 98,and/or at least one phenylalanine in any one of the positions 15, 46,48, 70 to 73, 75, 93, 95 and 98.

According to another preferred embodiment of the present invention atleast two amino acid residues in positions 15 to 17, 29 to 34, 85.4 to85.3, 92 to 94, 97 to 98 and/or 108 to 110 of a human or humanizedsingle domain antibody are modified.

The nucleic acid molecules encoding the modified immunoglobulins orimmunoglobulin variable domains (and always included throughout thewhole specification: immunoglobulins and immunoglobulin fragmentscomprising a modified immunoglobulin variable domain) may be cloned intohost cells, expressed and assayed for their binding specificities. Thesepractices are carried out using well-known procedures, and a variety ofmethods that may find use in the present invention are described inMolecular Cloning-A Laboratory Manual, 3.sup.rd Ed. (Maniatis, ColdSpring Harbor Laboratory Press, New York, 2001), and Current Protocolsin Molecular Biology (John Wiley & Sons). The nucleic acids that encodethe modified immunoglobulins or immunoglobulin variable domains of thepresent invention may be incorporated into an expression vector in orderto express said immunoglobulins. Expression vectors typically comprisean immunoglobulin operably linked—that is placed in a functionalrelationship—with control or regulatory sequences, selectable markers,any fusion partners, and/or additional elements. The modifiedimmunoglobulins of the present invention may be produced by culturing ahost cell transformed with nucleic acid, preferably an expressionvector, containing nucleic acid encoding the modified immunoglobulins,under the appropriate conditions to induce or cause expression of themodified immunoglobulins. The methods of introducing exogenous nucleicacid molecules into a host are well known in the art, and will vary withthe host used. Of course, also non-cellular or cell-free expressionsystems for the expression of modified immunoglobulins may be employed.

In a preferred embodiment of the present invention, the modifiedimmunoglobulins are purified or isolated after expression. Modifiedimmunoglobulins may be isolated or purified in a variety of ways knownto those skilled in the art. Standard purification methods includechromatographic techniques, electrophoretic, immunological,precipitation, dialysis, filtration, concentration, and chromatofocusingtechniques. Purification can often be enabled by a particular fusionpartner. For example, antibodies may be purified using glutathione resinif a GST fusion is employed, Ni²⁺-affinity chromatography if a His-tagis employed or immobilized anti-flag antibody if a flag-tag is used. Forgeneral guidance in suitable purification techniques, see e.g. Scopes,“Protein Purification: Principles and Practice”, 1994, 3^(rd) ed.,Springer-Science and Business Media Inc., NY or Roe, “ProteinPurification Techniques: A Practical Approach”, 2001, Oxford UniversityPress. Of course, it is also possible to express the modifiedimmunoglobulins according to the present invention on the surface of ahost, in particular on the surface of a bacterial, insect or yeast cellor on the surface of phages or viruses.

Modified immunoglobulins of the invention may be screened using avariety of methods, including but not limited to those that use in vitroassays, in vivo and cell-based assays, and selection technologies.Automation and high-throughput screening technologies may be utilized inthe screening procedures. Screening may employ the use of a fusionpartner or label, for example an enzyme, an immune label, isotopiclabel, or small molecule label such as a fluorescent or colorimetric dyeor a luminogenic molecule.

In a preferred embodiment, the functional and/or biophysical propertiesof the immunoglobulins are screened in an in vitro assay. In a preferredembodiment, the antibody is screened for functionality, for example itsability to catalyze a reaction or its binding specificity, crossreactivity and/or affinity to its target.

In another preferred embodiment, the favourable modified immunoglobulindomains may be selected in vivo, e.g. by introducing it into a cell oran organism. The specifically binding variants may be isolated eitherfrom body fluid such as blood or lymphatic liquid or from specificorgans, depending on the required properties of the modified domains.

Assays may employ a variety of detection methods including but notlimited to chromogenic, fluorescent, luminescent, or isotopic labels.

As is well known in the art, there are a variety of selectiontechnologies that may be used for the identification and isolation ofproteins with certain binding characteristics and affinities, including,for example, display technologies such as phage display, ribosomedisplay, cell surface display, and the like, as described below. Methodsfor production and screening of antibody variants are well known in theart. General methods for antibody molecular biology, expression,purification, and screening are described in Antibody Engineering,edited by Duebel & Kontermann, Springer-Verlag, Heidelberg, 2001; andHayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard &Georgiou, 2000, Annu Rev Biomed Eng 2:339-76.

As is known in the art, some screening methods select for favourablemembers of a library. The methods are herein referred to as “selectionmethods”, and these methods find use in the present invention forscreening modified immunoglobulins. When variant immunoglobulin variabledomain libraries are screened using a selection method, only thosemembers of a library that are favourable, that is which meet someselection criteria, are propagated, isolated, and/or observed. As willbe appreciated, because only the fittest variants are observed, suchmethods enable the screening of libraries that are larger than thosescreenable by methods that assay the fitness of library membersindividually. Selection is enabled by any method, technique, or fusionpartner that links, covalently or non-covalently, the phenotype ofimmunoglobulins with its genotype that is the function of an antibodywith the nucleic acid that encodes it. For example the use of phagedisplay as a selection method is enabled by the fusion of librarymembers to a phage coat protein (most frequently used is the filamentousbacteriophage gene III protein, however also other coat proteins such asprotein VIII, protein VII, protein VI and protein IX can be used). Inthis way, selection or isolation of modified immunoglobulins that meetsome criteria, for example binding affinity to the immunoglobulin'starget, also selects for or isolates the nucleic acid that encodes it.Once isolated, the gene or genes encoding modified immunoglobulins maythen be amplified. This process of isolation and amplification, referredto as panning, may be repeated, allowing favourable antibody variabledomain variants in the library to be enriched. Nucleic acid sequencingof the attached nucleic acid ultimately allows for gene identification.

A variety of selection methods are known in the art that may find use inthe present invention for screening immunoglobulin or immunoglobulinvariable domain libraries. These include but are not limited to phagedisplay (Phage display of peptides and antibodies: a laboratory manual,Kay et al., 1996, Academic Press, San Diego, Calif., 1996; Lowman etal., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science228:1315-1317) and its derivatives such as selective phage infection(Malmborg et al., 1997, J Mol Biol 273:544-551), selectively infectivephage (Krebber et al., 1997, J Mol Biol 268:619-630), and delayedinfectivity panning (Benhar et al., 2000, J Mol Biol 301:893-904), cellsurface display (Witrrup, 2001, Curr Opin Biotechnol, 12:395-399) suchas display on bacteria (Georgiou et al., 1997, Nat Biotechnol 15:29-34;Georgiou et al., 1993, Trends Biotechnol 11:6-10; Lee et al., 2000, NatBiotechnol 18:645-648; Jun et al., 1998, Nat Biotechnol 16:576-80),yeast (Boder & Wittrup, 2000, Methods Enzymol 328:430-44; Boder &Wittrup, 1997, Nat Biotechnol 15:553-557), and mammalian cells(Whitehorn et al., 1995, Bio/technology 13:1215-1219), as well as invitro display technologies (Amstutz et al., 2001, Curr Opin Biotechnol12:400-405) such as polysome display (Mattheakis et al., 1994, Proc NatlAcad Sci USA 91:9022-9026), ribosome display (Hanes et al., 1997, ProcNatl Acad Sci USA 94:4937-4942), mRNA display (Roberts & Szostak, 1997,Proc Natl Acad Sci USA 94:12297-12302; Nemoto et al., 1997, FEES Lett414:405-408), and ribosome-inactivation display system (Zhou et al.,2002, J Am Chem Soc 124, 538-543).

Other selection methods that may find use in the present inventioninclude methods that do not rely on display, such as in vivo methodsincluding but not limited to periplasmic expression and cytometricscreening (Chen et al., 2001, Nat Biotechnol 19:537-542), the antibodyfragment complementation assay (Johnsson & Varshaysky, 1994, Proc NatlAcad Sci USA 91:10340-10344; Pelletier et al., 1998, Proc Natl Acad SciUSA 95:12141-12146), and the yeast two hybrid screen (Fields & Song,1989, Nature 340:245-246) used in selection mode (Visintin et al., 1999,Proc Natl Acad Sci USA 96:11723-11728). In an alternate embodiment,selection is enabled by a fusion partner that binds to a specificsequence on the expression vector, thus linking covalently ornoncovalently the fusion partner and associated immunoglobulin librarymember with the nucleic acid that encodes them. For example, WO9308278describe such a fusion partner and technique that may find use in thepresent invention. In an alternative embodiment, in vivo selection canoccur if expression of the antibody imparts some growth, reproduction,or survival advantage to the cell.

Some selection methods are referred to as “directed evolution” methods.Those methods include the mating or breeding of favourable sequencesduring selection, sometimes with the incorporation of new mutations. Aswill be appreciated by those skilled in the art, directed evolutionmethods can facilitate identification of the most favourable sequencesin a plurality of polypeptides, and can increase the diversity ofsequences that are screened. A variety of directed evolution methods areknown in the art that may find use in the present invention forgenerating and screening antibody variable domain variants, includingbut not limited to DNA shuffling (PCT WO00/42561; PCT WO 01/70947), exonshuffling (Kolkman & Stemmer, 2001, Nat Biotechnol 19:423-428), familyshuffling (Crameri et al., 1998, Nature 391:288-291), selectivecombinatorial randomization (WO03012100, WO04018674A1), RandomChimeragenesis on Transient Templates (Coco et al., 2001, Nat Biotechnol19:354-359), molecular evolution by staggered extension process (StEP)in vitro recombination (Zhao et al., 1998, Nat Biotechnol 16:258-261;Shao et al., 1998, Nucleic Acids Res 26:681-683), exonuclease mediatedgene assembly (U.S. Pat. No. 6,352,842; U.S. Pat. No. 6,361,974), GeneSite Saturation Mutagenesis (U.S. Pat. No. 6,358,709), Gene Reassembly(U.S. Pat. No. 6,358,709), SCRATCHY (Lutz et al., 2001, Proc Natl AcadSci USA 98:11248-11253), DNA fragmentation methods (Kikuchi et al., Gene236:159-167), single-stranded DNA shuffling (Kikuchi et al., 2000, Gene243:133-137), and directed evolution antibody engineering technology(Applied Molecular Evolution) (U.S. Pat. No. 5,824,514; U.S. Pat. No.5,817,483; U.S. Pat. No. 5,814,476; U.S. Pat. No. 5,763,192; U.S. Pat.No. 5,723,323).

In a preferred embodiment, immunoglobulins or antibody variable domainvariants are screened using one or more cell-based or in vivo assays.For such assays, purified or non-purified modified immunoglobulins orimmunoglobulin variable domains are typically added exogenously suchthat cells are exposed to individual immunoglobulins or modifiedimmunoglobulin variable domains or pools of modified immunoglobulinvariable domains belonging to a library. These assays are typically, butnot always, based on the desired function of the immunoglobulin orimmunoglobulin variable domain; that is, the ability of the antibody orantibody variable domain modified according to the invention to bind toits target and to mediate some biochemical event, for example effectorfunction, ligand/receptor binding inhibition, apoptosis, and the like.Such assays often involve monitoring the response of cells to theantibody variable domain, for example cell survival, cell death, changein cellular morphology, or transcriptional activation such as cellularexpression of a natural gene or reporter gene. For example, such assaysmay measure the ability of variable immunoglobulin domain variants toelicit ADCC, ADCP or CDC. For some assays additional cells orcomponents, that is in addition to the target cells, may need to beadded, for example serum complement, or effector cells such asperipheral blood monocytes (PBMCs), NK cells, macrophages, and the like.Such additional cells may be from any organism, preferably humans, mice,rat, rabbit, and monkey. Immunoglobulins may cause apoptosis of certaincell lines expressing the target, or they may mediate attack on targetcells by immune cells which have been added to the assay. Methods formonitoring cell death or viability are known in the art, and include theuse of dyes, immunochemical, cytochemical, and radioactive reagents. Forexample, caspase staining assays may enable to measure apoptosis, anduptake or release of radioactive substrates or fluorescent dyes mayenable cell growth or activation to be monitored.

Alternatively, dead or damaged target cells may be monitored bymeasuring the release of one or more natural intracellular components,e.g. lactate dehydrogenase. Transcriptional activation may also serve asa method for assaying function in cell-based assays. In this case,response may be monitored by assaying for natural genes which may beupregulated, for example the release of certain interleukins may bemeasured, or alternatively readout may be via a reporter system.Cell-based assays may also involve the measure of morphological changesof cells as a response to the presence of modified immunoglobulinvariable domains. Cell types for such assays may be prokaryotic oreukaryotic, and a variety of cell lines that are known in the art may beemployed.

Alternatively, cell-based screens may be performed using cells that havebeen transformed or transfected with nucleic acids encoding the variantimmunoglobulin variable domains. In this case, antibody variable domainvariants of the invention are not added exogenously to the cells (e.g.Auf der Maur, 2004, Methods, 34:215-224). In another alternative method,the cell-based screen utilizes cell surface display. A fusion partnercan be employed that enables display of modified immunoglobulin variabledomains on the surface of cells (Witrrup, 2001, Curr Opin Biotechnol,12:395-399).

In a preferred embodiment, the immunogenicity of the modifiedimmunoglobulins may be changed and determined experimentally using oneor more immunological or cell based assays (e.g. Koren et al., 2002,Current Pharmaceutical Biotechnology 3:349-360; Chirino et al., 2004,Drug Discovery Today 9:82-90; Tangri et al., 2005, J. Immunol.174:3187-3196; Hermeling et al., 2004, Pharm. Res. 21:897-903). In apreferred embodiment, ex vivo T-cell activation assays are used toexperimentally quantitate immunogenicity. In this method,antigen-presenting cells and naive T-cells from matched donors arechallenged with a peptide or whole antibody or immunoglobulin ofinterest one or more times. T-cell activation can be detected using anumber of methods, e.g. by monitoring of cytokine release or measuringuptake of tritiated thymidine. In preferred embodiments, LUMINEXtechnology is used to measure cytokine release (e.g. de Jager et al.,Clin. Diagn. Lab. Immunol., 2003, 10:133-139) or interferon gammaproduction is monitored using Elispot assays (Schmittel et. al., 2000,J. Immunol. Meth., 24: 17-24).

The biological or functional properties of the modified immunoglobulinsor immunoglobulin variable domains of the present invention may becharacterized in cell, tissue, and whole organism experiments. As isknown in the art, drugs are often tested in animals, including but notlimited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in orderto measure a drug's efficacy for treatment against a disease or diseasemodel, or to measure a drug's pharmacokinetics, toxicity, and otherproperties. The animals may be referred to as disease models.Therapeutics are often tested in mice, including but not limited to nudemice, SCID mice, xenograft mice, and transgenic mice (including geneticknock-in and knock-out mutants). Such experimentation may providemeaningful data for determination of the potential of the polypeptidevariant to be used as a therapeutic. Any organism, preferably mammals,may be used for testing. Because of their genetic similarity to humans,monkeys can be suitable therapeutic models, and thus may be used to testthe efficacy, toxicity, pharmacokinetics, or other property of themodified immunoglobulins of the present invention. Testing drugcandidates in humans are mostly required for approval as therapeutics,and thus of course these experiments are contemplated. Thus the modifiedimmunoglobulins or immunoglobulin variable domains of the presentinvention may be tested in humans to determine their therapeuticefficacy, toxicity, immunogenicity, pharmacokinetics, pharmacodynamicsand/or other clinical properties.

The immunoglobulin according to the present invention may be used forany purpose known in the art for immunoglobulins but also enablesapplications which are depending on the combination of specificitiesintroduced by the present invention.

In one embodiment the antibody variant of the present invention is usedfor therapy or prophylaxis, for preparative or analytic use, as adiagnostic, an industrial compound or a research reagent, preferably atherapeutic. The antibody variant may find use in an antibodycomposition that is monoclonal, oligoclonal or polyclonal. In apreferred embodiment, the modified immunoglobulins or immunoglobulinvariable domains of the present invention are used to kill target cellsthat bear the target antigen, for example cancer cells. In an alternateembodiment, the modified immunoglobulins of the present invention areused to block, antagonize, or agonize the target antigen, for example byantagonizing a cytokine or cytokine receptor. In an alternatelypreferred embodiment, the modified immunoglobulins of the presentinvention are used to block, antagonize, or agonize the target antigenand kill the target cells that bear the target antigen.

In an alternately preferred embodiment, the modified immunoglobulins ofthe present invention are used to block, antagonize, or agonize growthfactors or growth factor receptors and kill the target cells that bearor need the target antigen. In an alternately preferred embodiment, themodified immunoglobulins of the present invention are used to block,antagonize, or agonize enzymes and substrate of enzymes. In anotheralternatively preferred embodiment, the modified immunoglobulin variabledomains of the present invention are used to neutralize infectiousagents such as viruses, small viruses, prions, bacteria or fungi.

The modified immunoglobulins or immunoglobulins variable domains of thepresent invention may be used for various therapeutic purposes. In apreferred embodiment, an antibody comprising the modified immunoglobulinor immunoglobulin variable domain is administered to a patient to treata specific disorder. A “patient” for the purposes of the presentinvention includes both, humans and other animals, preferably mammalsand most preferably humans. By “specific disorder” herein is meant adisorder that may be ameliorated by the administration of apharmaceutical composition comprising a modified immunoglobulin orimmunoglobulin variable domain of the present invention.

In one embodiment, a modified immunoglobulin or immunoglobulin variabledomain according to the present invention is the only therapeuticallyactive agent administered to a patient. Alternatively, the modifiedimmunoglobulin or immunoglobulin variable domain according the presentinvention is administered in combination with one or more othertherapeutic agents, including but not limited to cytotoxic agents,chemotherapeutic agents, cytokines, growth inhibitory agents,anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,cardioprotectants, or other therapeutic agents. The modifiedimmunoglobulin or immunoglobulin variable domain may be administeredconcomitantly with one or more other therapeutic regimens. For example,an antibody variant of the present invention may be formulated andadministered to the patient along with chemotherapy, radiation therapy,or both chemotherapy and radiation therapy. In one embodiment, themodified immunoglobulin or immunoglobulin variable domain of the presentinvention may be administered in conjunction with one or moreantibodies, which may or may not comprise an antibody variant of thepresent invention. In accordance with another embodiment of theinvention, the modified immunoglobulin or immunoglobulin variable domainof the present invention and one or more other anti-cancer therapies areemployed to treat cancer cells ex vivo. It is contemplated that such exvivo treatment may be useful in bone marrow transplantation andparticularly, autologous bone marrow transplantation. It is of coursecontemplated that the antibodies of the invention can be employed incombination with still other therapeutic techniques such as surgery.

A variety of other therapeutic agents may find use for administrationwith the modified immunoglobulins of the present invention. In oneembodiment, the modified immunoglobulin is administered with ananti-angiogenic agent, which is a compound that blocks, or interferes tosome degree with the development of blood vessels. The anti-angiogenicfactor may, for instance, be a small molecule or a protein, for examplean antibody, Fc fusion, or cytokine, that binds to a growth factor orgrowth factor receptor involved in promoting angiogenesis. The preferredanti-angiogenic factor herein is an antibody that, binds to VascularEndothelial Growth Factor (VEGF). In an alternate embodiment, themodified immunoglobulin is administered with a therapeutic agent thatinduces or enhances adaptive immune response, for example an antibodythat targets CTLA-4. In an alternate embodiment, the modifiedimmunoglobulin is administered with a tyrosine kinase inhibitor, whichis a molecule that inhibits to some extent tyrosine kinase activity of atyrosine kinase. In an alternate embodiment, the modifiedimmunoglobulins of the present invention are administered with acytokine. By “cytokine” as used herein is meant a generic term forproteins released by one cell population that act on another cell asintercellular mediators including chemokines.

Pharmaceutical compositions are contemplated wherein modifiedimmunoglobulins of the present invention and one or more therapeuticallyactive agents are formulated. Formulations of the polypeptide variantsof the present invention are prepared for storage by mixing saidmodified immunoglobulin or immunoglobulin variable domain having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's PharmaceuticalSciences, 1980, 16^(th) edition, Osol, A. Ed.,), in the form oflyophilized formulations or aqueous solutions. The formulations to beused for in vivo administration are preferably sterile. This is readilyaccomplished by filtration through sterile filtration membranes or othermethods. The modified immunoglobulins and other therapeutically activeagents disclosed herein may also be formulated as immunoliposomes,and/or entrapped in microcapsules.

Administration of the pharmaceutical composition comprising a modifiedimmunoglobulin or immunoglobulin variable domain of the presentinvention, preferably in the form of a sterile aqueous solution, may beperformed in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intranasally, intraotically,transdermally, topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary, vaginally,parenterally, rectally, or intraocularly.

Another aspect of the present invention relates to a method formanufacturing a molecule comprised of an immunoglobulin orimmunoglobulin variable domain or a pharmaceutical preparation thereofcomprising at least one modification in each of two structural loops orloop regions of said immunoglobulin or immunoglobulin variable domainand determining the binding of said molecule to an epitope of anantigen, wherein the unmodified molecule does not significantly bind tosaid epitope, comprising the steps of:

-   -   providing a nucleic acid encoding an immunoglobulin variable        domain comprising at least two structural loops or loop regions,    -   modifying at least one nucleotide residue in each of said        structural loops or loop regions,    -   transferring said modified nucleic acid in an expression system,    -   expressing said modified immunoglobulin,    -   contacting the expressed modified immunoglobulin with an        epitope,    -   determining whether said modified immunoglobulin binds to said        epitope, and    -   providing the modified immunoglobulin binding to said epitope        and optionally finishing it to a pharmaceutical preparation.

In particular the present invention relates to a method formanufacturing a multi-specific molecule binding specifically to at leastone first molecule or a pharmaceutical preparation thereof comprising atleast one modification in each of at least two structural loops or loopregions of an immunoglobulin or immunoglobulin variable domain anddetermining the specific binding of said at least two loops or loopregions to at least one second molecule selected from antigens. Theimmunoglobulin or immunoglobulin variable domain containing anunmodified structural loops or loop regions does not specifically bindto said at least one second molecule.

The method specifically comprises the steps of:

-   -   providing a nucleic acid encoding an immunoglobulin binding        specifically to at least one first molecule comprising at least        one structural loops or loop regions,    -   modifying at least one nucleotide residue of at least one of        said loops or loop regions encoded by said nucleic acid,    -   transferring said modified nucleic acid in an expression system,    -   expressing said modified immunoglobulin,    -   contacting the expressed modified immunoglobulin with said at        least one second molecule, and    -   determining whether said modified immunoglobulin binds        specifically to the second molecule and    -   providing the modified immunoglobulin binding specifically to        said at least one second molecule and optionally finishing it to        a pharmaceutical preparation.

The engineering of more than one binding site or at least twospecificities into a member of a specific binding pair is preferred(Kufer et al. (2004) Trends in Biotechnology vol. 22 pages 238-244).

Numerous attempts have been made to produce multi-specific, e.g.bispecific, monoclonal antibodies or antibody fragments. One problem inthe production of bispecific antibodies made of two differentpolypeptide chains (heavy and light chain) is the necessity to expressfour different chains (two heavy and two light chains) in one cellresulting in a number of various combinations of molecules which have tobe separated from the desired bispecific molecule in the mixture. Due totheir similarity the separation of these molecules is difficult andexpensive. A number of techniques have been employed to minimize theoccurrence of such unwanted pairings (Carter (2001) Journal ofImmunological Methods, vol 248, pages 7-15)

One solution to the problem is the production of one polypeptide chainwith two specificities, like e.g. two scFvs linked to each other or theproduction of so-called diabodies. Such molecules have been shown to befar away from the fold of a natural molecule and are notoriouslydifficult to produce (LeGall et al. (2004) Protein Engineering, Design &Selection vol 17 pages 357-366).

According to a preferred embodiment of the present invention theexpression system comprises a vector. Any expression vector known in theart may be used for this purpose as appropriate.

The modified immunoglobulin is preferably expressed in a host,preferably in a bacterium, in yeast, in a plant cell, in an insect cell,in an animal cell or mammalian cell or in an organ of a plant or animalor in a complete plant or animal.

A wide variety of appropriate host cells may be used to express themodified polypeptide of the invention, including but not limited tomammalian cells (animal cells) plant cells, bacteria (e.g. Bacillussubtilis, Escherichia coli), insect cells, and yeast (e.g. Pichiapastoris, Saccharomyces cerevisiae). For example, a variety of celllines that may find use in the present invention are described in theATCC cell line catalog, available from the American Type CultureCollection. Furthermore, also plants and animals may be used as hostsfor the expression of the immunoglobulin according to the presentinvention. The expression as well as the transfection vectors orcassettes may be selected according to the host used.

Of course also non-cellular or cell-free protein expression systems maybe used. In vitro transcription/translation protein expressionplatforms, that produce sufficient amounts of protein offer manyadvantages of a cell-free protein expression, eliminating the need forlaborious up- and down-stream steps (e.g. host cell transformation,culturing, or lysis) typically associated with cell-based expressionsystems.

Another problem of the current design of bispecific antibodies is thefact that even if the parent antibodies are bivalently binding to theirrespective binding partner (e.g. IgG), the resulting bispecific antibodyis monovalent for each of the respective binding partner.

The preferred multi-specific molecules of the present invention solvethese problems:

Expression of a bispecific molecule as one polypeptide chain is possible(a modified immunoglobulin variable domain with two bindingspecificities, see example section), which is easier to accomplish thanthe expression of two antibody polypeptide chains (Cabilly et al. Proc.Natl. Acad. Sci. USA 81:3273-3277 (1984).

It can also be produced as an antibody like molecule (i.e. made of 2polypeptide chains). Due to the fact that the second specificity islocated in the non-CDR part of the variable domains there is no need fortwo different heavy chains or different light chains. Thus, there is nopossibility of wrong pairing of the two chains.

An antibody of the present invention may consist of a heavy chain and alight chain, which form together a variable CDR loop region binding to aspecific binding partner, i.e. a specific CDR loop conformation, and thesecond specificity may be formed by modified structural loops or loopregions contained in the immunoglobulin molecule, e.g. the structuralloops of either the heavy chain or the light chain variable domain,while maintaining the specific CDR loop conformation. The binding sitemay also be formed by at least one or more than one non-CDR loops on twovariable domains (e.g. a heavy chain variable domain and a light chainvariable domain which may be structurally neighboured).

The modified antibody may be a complete antibody or an antibody fragment(e.g. Fab, scFv, Fv, minibody, dAb) comprising at least oneimmunoglobulin variable domain and modifications in the structural loopsor loop regions, or derivatives thereof.

It may bind mono- or multivalently to binding partners or even withdifferent valency for the different binding partners, depending on thedesign. For example, an Fab fragment or, equivalently an scFv may beengineered in such a way that the structural loops of the VH and the VLdomains are separately engineered to bind to the same epitope as thebinding site formed by the CDRs, resulting in a trivalent Fab fragmentor scFv respectively. In another embodiment, a complete immunoglobulincontaining the same engineered VH and VL domains will bind hexavalentlyto its target epitope. If for example the natural binding site formed bythe CDRs recognizes a different target epitope than the engineered Vhand Vl domains then the resulting Fab fragment or scFv will bindmonovalently to the first target, and bivalently to the second targetwhich is bound independantly by the modified structural loops of the VHand the VL domain respectively. This modular design principle can beapplied in numerous different ways as will be obvious to those skilledin the art.

As there are a number of various structural loops available forselection and design of a specific binding site in the non-CDR regionsof heavy and light domains it is possible to design antibody derivativeswith even more than two specificities. For example, VH and VL domainsrecognizing a first target by their CDRs can be engineered separately tobind specifically to different (second and third) targets throughinteractions mediated by the modified structural loops. Thus, atrispecific Fab fragment or scFv, which binds monovalenty to each of itstarget can be generated. If the modified variable domains of this Fabare engineered in the form of a full size IgG, an engineered IgG isgenerated which is trispecific and, to each if its three specificities,binds bivalently.

The specific binding domains within one polypeptide chain may beconnected with or without a peptide linker.

Some antibody classes can be regarded as multi-specific, in particularbispecific, by nature: They bind to an antigen (which is typically e.g.either a foreign structure or a cancer associated structure) with thevariable region and they bind to Fc-effector molecules with the Fc part(e.g. Fc receptors on various immune cells or complement protein) thusenabling effects such as ADCC, ADCP or CDC.

The Fc-effector molecules are bound by the Fc-part of an immunoglobulinmolecule (for IgG1 it consists of domains CH2 and CH3) and a number ofmethods have been described to optimize effector function by improvementof binding of the Fc-part of an antibody molecule either byglycoengineering techniques (U.S. Pat. No. 6,602,684) or by proteinengineering either directly at the Fc (US 2005/0054832) or indirectly byengineering outside the Fc (US 2005/02444403). Both, binding of the Fcregion to Fc receptor and/or binding to complement proteins such Cq1 hasbeen altered by such techniques. Usually one tries to improve thebinding affinity to such Fc-effector molecules as this correlates withimproved effector functions.

With the current invention it is possible to design an antibody bindingto Fc-effector molecules outside the natural Fc binding region. Modifiedloops in antibody variable domains other than the loops involved in“natural” Fc-effector molecule binding can be selected from a library ofmodified loop structures or designed to bind to one or more Fc-effectormolecules. An antibody with such additional Fc-effector molecule bindingsites would either have stronger avidity to a certain Fc-effectormolecule or effector-cell displaying an Fc-effector molecule andtherefore may have an even stronger effect than glycoengineeredantibodies or otherwise improved Fc regions.

Antibody fragments have certain advantages as compared to wholeantibodies. Fragments have usually good biodistribution properties andcan more easily be produced. However, most of the antibody fragmentdesigns lack effector functions and have short in vivo half life(Holliger P, et al. Nat Biotechnol. (2005) 23:1126-36.). Neither CH1 norCκ or Cλ domains are able to mediate effector functions, which is thereason why Fab molecules usually do not show ADCC, ADCP or CDC.

WO 02/44215 describes binding molecules which consist of the antigenbinding site of an antibody and a peptide binding Fc-effector molecules.In such a way an antibody fragment displaying effector functions can beconstructed. The peptide is being incorporated into the binding moleculeat a position that does neither destroy the antigen binding nor theability of the peptide to bind to an Fc-effector molecule.

According to the present invention however, the binding to Fc-effectormolecules may be performed with modified immunoglobulins orimmunoglobulin variable domains which have been selected for Fc-effectormolecule binding from libraries of two, three or four randomizedstructural loop sequences within a fixed scaffold of an immunoglobulinor immunoglobulin variable domain. Therefore, it is possible to selectfor specific loop sequences which would not bind to Fc-effectormolecules if isolated from the Ig-domain scaffold. The polypeptidesresulting from the present invention may therefore preferably consist ofmore than 100 amino acids and may comprise one or more immunoglobulinvariable domains.

In order to select for potential effector function of such variabledomains according to the present invention, antibody- or antibodyfragment libraries comprising mutant variable domains may be selectedfor binding to Fc-receptors and/or complement factors such as C1q.Fcgamma receptors for selection may be provided either on the surface ofcells expressing naturally the respective receptors or by expression andpurification of the extracellular part of the respective receptor. IFN-gstimulated U937 cells (CRL-1503, American Type Culture Collection) canbe used as target cells for the isolation of phage displayed modifiedimmunoglobulin variable domains that bind specifically to thehigh-affinity IgG receptor, FcgammaRI (Berntzen et al., 2006, ProteinEng Des Sel. 19(3):121-8). Binding to the Fc receptor can be tested forby FACS using U937 cells as target, which cells are stained specificallywith selected modified immunoglobulin variable domains. Furthermore, theextracellular domains of human Fcgamma receptors can be cloned andexpressed as soluble proteins or fusion proteins and used for analysisof the specific binding of potential binding partners (e.g. as inBerntzen et al., 2005, J Immunol Methods. 298(1-2):93-104). Theidentification and characterisation of modified immunoglobulin variabledomains specifically binding to complement factor C1q can be performedessentially similarily (e.g. as in Lauvrak et al. 1997 Biol Chem.378(12):1509-19).

In order to increase in vivo half life of a molecule consisting of orcontaining such a variable domain binding to FcRn, or serum albumin maybe selected for with libraries of mutant variable domains according tothe present invention. Those modified structural loops responsible forextending the half life of a molecule through its specific binding toserum proteins or complement proteins can be used as isolated structuralloops or in the context of an immunoglobulin or parts thereof, forcombination with those molecules that are to be designed as moleculeswith increased half life in vivo.

FcRn receptors or other cell receptors for selection may be providedeither on the surface of cells expressing naturally the respectivereceptors or by expression and purification of the extracellular part ofthe respective receptor. For the purpose of this invention a firstscreening on FcRn may select for mutant variable domains (or moleculescomprising such mutant variable domains) which can further be tested invitro and even further characterized in FACS experiments by binding tocells expressing FcRn receptor. Screening and selection may alsoconsider pH dependencies in binding to FcRn (as described in PCTWO02/060919; PCT WO97/34631). It can be further characterized byaffinity ranking of binding to various recombinant FcRn, isoforms andallotypes e.g with surface plasmon resonance techniques (e.g. as inDall' Acqua et al. Journal of Immunology, 2002, 169: 5171-5180).

The modified immunoglobulin according to the invention may comprise aheavy and/or light chain, or parts thereof, and at least one variabledomain.

The immunoglobulin according to the present invention comprisespreferably at least one constant and/or at least one variable domain ofthe immunoglobulin, or a part thereof.

A variable domain usually is considered an immunoglobulin fold unit ofthe variable part of an immunoglobulin, also referred to as a domain ofthe variable region (e.g. VH, Vk, Vl, Vd)

Another preferred immunoglobulin according to the invention consists ofa variable domain of a heavy or light chain, or a part thereof, with atleast two structural loops or loop regions, and is characterised in thatsaid at least two structural loops or loop regions comprise at least twoamino acid modifications forming at least two modified structural loopsor loop regions, wherein said at least two modified structural loops orloop regions bind specifically to at least one epitope of an antigen. Insuch a preferred immunoglobulin according to the invention the at leasttwo amino acid modifications may be located in one or two structuralloops or loop regions or at one or two structural loops so to make up abinding site for an antigen.

According to a preferred embodiment of the present invention thespecific binding of the modified polypeptide to a molecule is determinedby a binding assay selected from the group consisting of immunologicalassays, preferably enzyme linked immunosorbent assays (ELISA), surfaceplasmon resonance assays, saturation transfer difference nuclearmagnetic resonance spectroscopy, transfer NOE (trNOE) nuclear magneticresonance spectroscopy, competitive assays, tissue binding assays, livecell binding assays and cellular extract assays.

Binding assays can be carried out using a variety of methods known inthe art, including but not limited to FRET (Fluorescence ResonanceEnergy Transfer) and BRET (Bioluminescence Resonance EnergyTransfer)-based assays, Amplified Luminescent Proximity HomogeneousAssay, Scintillation Proximity Assay, ELISA (Enzyme-Linked ImmunosorbentAssay), SPR (Surface Plasmon Resonance), isothermal titrationcalorimetry, differential scanning calorimetry, gel electrophoresis, andchromatography including gel filtration.

The modified polypeptide of the invention is preferably conjugated to alabel selected from the group consisting of organic molecules, enzymelabels, radioactive labels, colored labels, fluorescent labels,chromogenic labels, luminescent labels, haptens, digoxigenin, biotin,metal complexes, metals, colloidal gold and mixtures thereof.

Modified polypeptides conjugated to labels as specified above may beused, for instance, in diagnostic methods.

The modified immunoglobulin may be conjugated to other molecules whichallow the simple detection of said conjugate in, for instance, bindingassays (e.g. ELISA) and binding studies.

Another aspect of the present invention relates to a polypeptidecomprising a variable domain of a light or heavy chain or combinationsthereof, with at least two loops or loop regions, characterised in thatsaid at least two structural loops or loop regions each comprise atleast one amino acid modification forming at least two modifiedstructural loops or loop regions, wherein said at least two modifiedstructural loops or loop regions bind specifically to at least oneepitope of an antigen.

It is preferred to combine molecularly at least one modified antibodyvariable domain (=binding to the specific partner via the non-variablesequences or structural loops) with at least one other binding moleculewhich can be an antibody, antibody fragment, a soluble receptor, aligand or another modified antibody domain.

The other binding molecule combined with the at least one modifiedantibody variable domain of the invention is selected from the groupconsisting of proteinaceous molecules, nucleic acids, and carbohydrates.

The structural loops or loop regions of the modified immunoglobulinaccording to the invention may specifically bind to any kind of bindingmolecules, in particular to proteinaceous molecules, proteins, peptides,polypeptides, nucleic acids, glycans, carbohydrates, lipids, small andlarge organic molecules, inorganic molecules. Of course, the modifiedimmunoglobulin according to the invention may comprise at least twoloops or loop regions whereby each of the loops or loop regions mayspecifically bind to different molecules or epitopes.

Another aspect of the present invention relates to the use of animmunoglobulin or immunoglobulin variable domain according to thepresent invention or obtainable by a method according to the presentinvention for the preparation of a vaccine for active immunization.Hereby the immunoglobulin is either used as antigenic drug substance toformulate a vaccine or used for fishing or capturing antigenicstructures for use in a vaccine formulation.

Another aspect of the present invention relates to the use of animmunoglobulin according to the present invention or obtainable by amethod according to the present invention for the preparation of alibrary of polypeptides comprising modified immunoglobulin variabledomains.

Yet another aspect of the present invention relates to a method forspecifically binding and/or detecting a target molecule comprising thesteps of:

-   -   (a) contacting a molecule comprising a modified immunoglobulin        or immunoglobulin variable domain according to the present        invention or a molecule comprising a modified immunoglobulin        variable domain obtainable by a method according to the present        invention with a test sample containing or suspected to contain        said target molecule, and optionally    -   (b) detecting the potential formation of a specific        immunoglobulin/molecule or immunoglobulin variable        domain/molecule complex.

A preferred method according to the invention is for specificallybinding and/or detecting a molecule comprising the steps of:

-   -   (a) contacting a library of modified immunoglobulins or a        modified immunoglobulin according to the present invention with        a test sample containing said molecule, and optionally    -   (b) detecting the potential formation of a specific        immunoglobulin/molecule complex.

Test samples may be human or animal sample, such as blood samples orother body fluids and cell suspensions, which sample possibly contains atarget molecule to be specifically bound by immunoglobulins forcapturing and/or detecting purposes.

Another aspect of the present invention relates to a method forspecifically isolating a target molecule comprising the steps of:

-   -   (a) contacting a molecule comprising a modified immunoglobulin        or immunoglobulin variable domain according to the present        invention or a molecule comprising a modified immunoglobulin        variable domain obtainable by a method according to the present        invention with a sample containing said target molecule,    -   (b) separating the specific immunoglobulin variable        domain/target molecule complex formed, and    -   (c) optionally isolating the target molecule from said complex.

A preferred method according to the invention is for specificallyisolating a modified immunoglobulin binding to a molecule comprising thesteps of:

-   -   (a) contacting a library of modified immunoglobulins according        to the present invention with a sample containing said molecule,    -   (b) separating the specific modified immunoglobulin/molecule        complex formed, and    -   (c) optionally isolating the modified immunoglobulin from said        complex.

Those samples are usually considered sources for preparative isolatingthose molecules, for instance complex natural sources, like animal,human or plant sources or microbial derived sources or cell suspensionsand cultures.

The immunoglobulins or immunoglobulin variable domains according to thepresent invention may be used to isolate specifically target moleculesfrom a sample. If multi-specific immunoglobulins or immunoglobulinvariable domains are used more than one target molecule may be isolatedfrom a sample. It is especially advantageous using immunoglobulins ormodified immunoglobulin variable domains in such methods because itallows, e.g., to generate a matrix having a homogeneous surface withdefined amounts of binding partners (i.e. modified immunoglobulinvariable domains) immobilised thereon which are able to bind to thetarget molecules to be isolated. In contrast thereto, if mono-specificbinding partners are used no homogeneous matrix can be generated becausethe single binding partners do not bind with the same efficiency to thematrix.

Another aspect of the present invention relates to a method fortargeting a compound to a target comprising the steps of:

-   -   (a) contacting a molecule comprising a modified immunoglobulin        variable domain according to the present invention or a molecule        comprising a modified immunoglobulin variable domain obtainable        by a method according to the present invention capable to        specifically bind to said compound,    -   (b) delivering the molecule comprising an immunoglobulin        variable domain/compound complex to the target.

Modified immunoglobulins or immunoglobulin variable domains according tothe present invention may be used to deliver at least one compound boundto the CDRs by a specific CDR loop conformation to a target by bindingto the modified structural loop region. Such immunoglobulins may be usedto target therapeutic substances to a preferred site of action in thecourse of the treatment of a disease.

Another aspect of the present invention relates to a molecule librarycomprising, expressing or encoding an immunoglobulin or immunoglobulinvariable domain according to the present invention or obtainable by themethod according to the present invention.

The preferred library of immunoglobulins or immunoglobulin variabledomains according to the invention comprises at least 10 immunoglobulinsor immunoglobulin variable domains, preferably 100, more preferably1000, more preferably 10000, more preferably 100000, most preferablymore than 1000000 variant immunoglobulins or variable domains, with amodification in at least two structural loops or loop regions.

Usually libraries according to the invention comprise at least 10 fusionproteins or binding agents, preferably at least 100, more preferred atleast 1000, more preferred at least 10⁴, more preferred at least 10⁵,more preferred at least 10⁶, more preferred at least 10⁷, more preferredat least 10⁸, more preferred at least 10⁹, more preferred at least 10¹⁰,more preferred at least 10¹¹, up to 10¹², in cases of ribosomal displayeven higher number are feasible.

It turned out that most preferred members of a library have mutations ofat least 4, or even at least 5 or 6 amino acid positions in at least twostructural loops or loop regions. Thus, the particularly preferredlibraries according to the invention consist of members that havemutations of at least 2, 3 or 4 amino acid positions in at least twostructural loops.

A library according to the invention may also comprises or consist ofone of or a mixture of immunoglobulin variable domains selected from thegroup of VH, Vkappa, Vlambda and VHH, as suitable for the purpose ofdefining binding partners for commercial reasons.

Preferred methods for constructing said library can be found above andin the examples. The library according to the present invention may beused to identify immunoglobulins or immunoglobulin variable domainsbinding to a distinct molecule.

In particular the present invention relates to the use of a proteinlibrary of polypeptides comprising an immunoglobulins or immunoglobulinvariable domain according to the present invention or obtainable by themethod according to the present invention for the design ofimmunoglobulin derivatives. An existing immunoglobulin can be changed tointroduce antigen binding sites into an immunoglobulin or a variabledomain by using a protein library of the respective immunoglobulin orvariable domain of at least 10, preferably 100, more preferably 1000,more preferably 10000, more preferably 100000, most preferably more than1000000 immunoglobulins or variant variable domains each with at leasttwo modified structural loops. The library is then screened for bindingto the specific antigen. After molecular characterization for thedesired properties the selected immunoglobulin or variable domain iscloned into the original immunoglobulin by genetic engineeringtechniques so that it replaces the wild type region. Alternatively, onlythe DNA coding for the modified loops or coding for the mutated aminoacids may be exchanged to obtain an immunoglobulin with the additionalbinding site for the specific antigen. Alternatively, the modificationin the structural loops of the variable domains may be performed withthe variable domain in its natural context, e.g. in the form of a Fab,scFv, or complete immunoglobulin molecule. Except when single domainimmunoglobulins, a chain of single immunoglobulin domains or singlechain immunoglobulins, such as scFv or unibodies (monovalentimmunoglobulin fragments) are produced, usually the immunoglobulinaccording to the invention is provided as a dimer, preferably as aheterodimer.

The choice of the site for the mutated, antigen-specific structural loopis dependent on the structure of the original immunoglobulin and on thepurpose of the additional binding site. If, for example, the original orparent immunoglobulin is a Fab or scFv, modification of at least twostructural loops in the variable domains of the light chain and/or theheavy chain is possible, but also modification of at least twostructural loops in the CH1/CL is preferred to produce an immunoglobulinaccording to the invention. Thus, the Fab molecule according to theinvention may contain the new binding site through a modified loopregion in the CH1 and or the CL domain. In this context it is usually ofprimary importance to maintain the specific CDR loop conformation andnatural binding properties of the parent immunoglobulin, scFv or Fab.

To generate a library one may prepare libraries of mutant originalmolecules which have mutations in two ore more structural loops of oneor more variable domains. The selection with complete mutated originalmolecules may have some advantages as the selection for antigen bindingwith a modified structural loop will deliver the sterically advantageousmodifications. For example, if the complete molecule is a scFv, it maybe advantageous to screen the library of mutated original scFv forbinding to an antigen, followed by screening the specific binders forbinding to the antigen which is recognized by the CDR loops (originalspecificity). In an alternative selection procedure the original—thefirst—antigen may be bound to the CDR-loops during the screening forbinding to an antigen with the modified structural loops. Thissimultaneous screening may allow for rescuing of clones that would belost during a sequential selection procedure if the binding to theantigen is influenced by the binding to the first antigen.

A preferred embodiment of the invention is a library of variantimmunoglobulins containing or consisting of variable domains with atleast one variant amino acid position in each of at least two of thestructural loops. The library may comprise immunoglobulin domains of theheavy and the light chain or mixtures and molecular combinationsthereof.

Another preferred embodiment is a library containing or consisting ofVHH domains or humanized forms of such camelid domains with at least onevariant amino acid position in each of at least two structural loops orloop regions.

Another preferred embodiment of the invention is a library containing orconsisting of single chain antibodies, such as a scFv library with atleast one variant amino acid position in each of at least two of thestructural loops or loop regions of any of the variable domains of thesingle chain antibody or scFv.

Another preferred embodiment of the invention is a diabody librarycontaining or consisting of at least one variant amino acid position ineach of at least two of the structural loops or loop regions of any ofthe variable domains of the diabody.

Another preferred embodiment of the invention is a minibody librarycontaining or consisting of at least one variant amino acid position ineach of at least two of the structural loops or loop regions of any ofthe variable domains of the minibody.

Yet another preferred embodiment of the invention is a Fab librarycontaining or consisting of at least one variant amino acid position ineach of at least two of the structural loops or loop regions of any ofthe variable domains of the Fab.

Yet another preferred embodiment of the invention is an antibody or IgGlibrary, preferably a human antibody library, containing or consistingof at least one variant amino acid position in each of at least two ofthe structural loops or loop regions of any of the variable domains ofthe antibody or IgG domains.

The size requirement (i.e. the number of variants) of a libraryaccording to the invention, comprising differently mutatedimmunoglobulins or immunoglobulin variable domains or fusion moleculesof mutated variable antibody domains is dependent on the task. Ingeneral, a library to generate an antigen binding site de novo needs tobe larger than a library used to further modify an already existingengineered antigen binding site formed by a modified structural loop orloop region (e.g. for enhancing affinity or changing fine specificity tothe antigen).

The present invention also relates to a polypeptide library or a nucleicacid library comprising a plurality of polypeptides comprisingimmunoglobulins or immunoglobulin variable domains or at least twostructural loops or loop regions contained in a minidomain, or nucleicacid molecules encoding the same. The library contains members withdifferent modifications, wherein the plurality is defined by themodifications in the at least two structural loops or loop regions. Thenucleic acid library preferably includes at least 10 different members(with at least two potential amino acid modifications) and morepreferably includes at least 100, more preferably 1000 or 10000different members (e.g. designed by randomisation strategies orcombinatory techniques). Even more diversified individual membernumbers, such as at least 1000000 or at least 10000000 are alsopreferred, more preferred at least 10⁸, more preferred at least 10⁹,more preferred at least 10¹⁰, more preferred at least 10¹¹, up to 10¹²,in cases of ribosomal display even higher number are feasible.

A further aspect of the invention is the combination of two differentimmunoglobulins or immunoglobulin variable domains selected from atleast two libraries according to the invention in order to generatemultispecific immunoglobulins. These selected specific immunoglobulinvariable domains may be combined with each other and with othermolecules, similar to building blocks, to design the optimal arrangementof the domains to get the desired properties such as combinations ofspecificities and/or valencies.

Furthermore, one or more modified immunoglobulins or immunoglobulinvariable domains according to the invention may be introduced at variousor all the different sites of a protein without destruction of thestructure of the protein. By such a “domain shuffling” technique newlibraries are created which can again be selected for the desiredproperties.

The preferred library contains immunoglobulins or immunoglobulinvariable domains according to the invention or derivatives thereof.

A preferred embodiment of the present invention is a binding moleculefor an antigen (antigen binding molecule) comprising at least oneimmunoglobulin or immunoglobulin variable domain and at least twostructural loops or loop regions being modified according to the presentinvention to bind to the antigen, wherein said binding molecule has norelevant and/or specific binding activity with its CDR-loops. It maycomprise other parts useable for antibody activities (e.g. such asnatural or modified effector regions (sequences); however, it lacks the“natural” binding region of antibodies, i.e. active CDR-loops in theirnaturally occurring position. These antigen binding molecules accordingto the present invention have the advantages described above for thepresent molecules, yet without the specific binding activity ofantibodies; however with a newly introduced specific binding activity inthe structural loop or loop region.

Also for the antigen binding molecules according to the presentinvention it is preferred that the new antigen binding sites in thestructural loops are introduced by randomising technologies, i.e. bymodifying one or more amino acid residues of at least two structuralloops by randomisation techniques or by introducing randomly generatedinserts into such structural loops. Alternatively preferred is the useof combinatorial approaches.

According to another aspect, the present invention relates to a modifiedimmunoglobulin having an antigen binding site foreign to the unmodifiedimmunoglobulin and incorporated in one, two, three or more structuralloops of the immunoglobulin or immunoglobulin variable domain. In thiscontect the term “foreign” means that the antigen binding site is notnaturally formed by the specific structural loop or loop region of thevariable immunoglobulin domain.

According to yet another aspect, the present invention relates to amodified immunoglobulin having an antigen binding site foreign to theunmodified immunoglobulin and incorporated in one, two, three or morestructural loops of the variable domain, wherein said modifiedimmunoglobulin binds to said antigen with an affinity of at least 10³mol⁻¹, at least 10⁴ mol⁻¹, at least 10⁵ mol⁻¹, at least 10⁶ mol⁻¹, atleast 10⁷ mol⁻¹, at least 10⁸ mol⁻¹, or at least 10⁹ mol⁻¹.

Usually binders with medium or high affinity are provided according tothe invention. Those with medium affinity preferably exert adissociation rate Kd in the range of 10⁻⁵ to 10⁻⁷, those with highaffinity have a proven Kd in the range of 10⁻⁸ to 10⁻¹⁰, those having aKd less than 10⁻⁹ being most preferred as a high affinity binder. Insome cases it will be appropriate to select binders with even lower Kd,e.g. less than 10⁻¹¹, usually as low as 10⁻¹².

Preferred immunoglobulins or immunoglobulin variable domains accordingto the present invention comprise at least two antigen binding sites,the first site binding to a first epitope, and the second site bindingto a second epitope.

According to a preferred embodiment, the present immunoglobulin orimmunoglobulin variable domain comprises at least three loops or loopregions, the first loop or loop region binding to a first epitope, andthe second and third loop or loop region binding to a second epitope.Either the at least first or at least second and third loop or loopregion or both may contain a structural loop. The immunoglobulin orimmunoglobulin variable domains according to the present inventionsinclude the fragments thereof known in the art to be functional whichcontain the essential elements according to the present invention: thestructural loops or loop regions modified according to the presentinvention.

Preferably, the immunoglobulin according to the present invention iscomposed of at least two immunoglobulin domains, or a part thereofincluding a minidomain, and each domain contains at least one antigenbinding site. One of the preferred pairs of immunoglobulin domains is aCL/CH1 pair, which may be engineered in the structural loop region thatis located at the C-terminus of the CL/CH1 pair. Thereby one or two newbinding sites are engineered. Upon selection of the specific CL/CH1binding domain pair, it may be recombined with the variable domains VLand VH to obtain a Fab molecule according tonthe invention with one“natural” binding site in the CDR region and one or two additionalbinding sites opposite thereto, i.e. in the C-terminal structural loopregion of the CH1/CL domains.

Thus, the immunoglobulin according to the invention may be obtained bymodifying an immunoglobulin parent molecule that contains the variabledomain. Alternatively, a constant immunoglobulin domain may beengineered to obtain a binding site in the structural loop region, whichdomain can then be used as a building block to produce combinations withvariable immunoglobulin domains and optionally with other constantdomains, resulting in an immunoglobulin according to the invention thatcontains both a variable domain and a new binding site formed bystructural loops or structural loop regions.

According to such preferred embodiment of the invention there isprovided an immunoglobulin, which comprises at least one domain of thevariable region of an immunoglobulin and at least one domain of theconstant region of an immunoglobulin; for example, a variable domain,which is modified in at least two structural loops linked to a CH1domain.

Another aspect of the present invention relates to a kit of bindingpartners containing

-   -   (a) a polypeptide comprising a modified immunoglobulin variable        domain having an antigen binding site incorporated in two or        more structural loops, and    -   (b) a binding molecule containing an epitope of said antigen.

Preferably a kit of binding partners according to the invention iscontaining

-   -   (a) a library of modified immunoglobulins according to the        present invention, and    -   (b) a binding molecule containing an epitope of an antigen.

Such a binding molecule of this kit according to the present inventionmay be used for selecting and distinguishing a native or modifiedimmunoglobulin according to the present invention in a sample or from alibrary. It may further be used for identifying the binding specificityof polypeptides comprising a modified immunoglobulin or immunoglobulinvariable domain according to the present invention. By using the bindingmolecule of this kit according to the present invention, the potency ofthe modified polypeptide according to the present invention may bedetermined.

Potency as defined here is the binding property of the modified moleculeof the invention to its antigen. The binding can be determinedquantitatively and/or qualitatively in terms of specificity and/oraffinity and/or avidity by assay methods as known in the art for qualitycontrol purposes.

Moreover, the binding molecule of a kit according to the presentinvention may be used for selecting the polypeptide comprising amodified immunoglobulin or immunoglobulin variable domain according tothe present invention from a library as specified above, preferablyconsisting of at least 10, preferably at least 100, more preferably atleast 1000, more preferred at least 10000, especially at least 100000polypeptides with different modifications in the structural loops.

The present invention is further illustrated by the following examples.

EXAMPLE 1 Design of the VHH Library

The crystal stucture of the camel VHH domain D2-L24 in complex with HenEgg White Lysozyme, which is published in the Brookhaven Database asentry 1ZVH.pdb was used to aid in the design of the mutated VHH domain.The sequence of chain A of the structure file 1ZVH.pdb is given in SEQID No. 1.

SEQ ID No. 1  PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKAAA

The sequence which was used as the basis for construction of the VHHlibrary is the sequence of the anti-TNF-alpha VHH domain from patentWO04041862A2¹, clone 3E and is given in SEQ ID No. 2.

SEQ ID No. 2 ccatggcccc ccgagaacca caggtgtaca ccctgcccccatcccgtgac gagctcnnsn nsnnscaagt cagcctgacctgcctggtca aaggcttcta tcccagcgac atcgccgtggagtgggagag caatgggcagccggagaaca actacaagaccacgcctccc gtgctggact ccgacggctc cttcttcctctacagcaagc ttaccgtgnn snnsnnsagg tggnnsnnsgggaacgtctt ctcatgctcc gtgatgcatg aggctctgcacaaccactac acacagaaga gcctctccct gtctccgggt aaagcggccg ca

After detailed analysis of the structure of 1ZVH.pdb and by visualinspection of the residues forming the loops which connect the betastrands, it was decided to randomize residues 13, 15, (i.e. in the loopbetween beta-strands A and B) 89, 90, 92 and 93 (i.e. in the loopbetween beta-strands E and F) of SEQ ID No. 2 for the generation of thelibrary. In addition, three randomized positions were decided to beinserted between residues 14 and 15, and three randomized positions weredecided to be inserted between residues 92 and 93 of SEQ ID No. 2.

EXAMPLE 2 Construction of the VHH Library

The engineered gene coding for the VHH sequence is produced in the formof a synthetic gene by PCR assembly. The sequence and its translationare shown in FIG. 2. Amino acid residues to be randomized for libraryconstruction are underlined. Restriction sites for cloning are includedas follows, and are underlined in the nucleotide sequence as shown inFIG. 2: NcoI, BglII and NotI.

The amino acid sequence encoded by the synthetic gene is given in SEQID. No.3.

SEQ ID No. 3 MAPREPQVYTLPPSRDELXXXQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVXXXRWXXGNVFSCSVMHEALHNHYTQKS LSLSPGKAAA

The first two residues and the last two residues are caused by therestriction sites used for cloning. The nucleotide sequence coding forSEQ ID No. 3 is given in SEQ ID No. 4. SEQ ID No.4: cttgccatggccccccgaga accacaggtg tac

The first two codons and the last two codons are caused by therestriction sites used for cloning.

The oligonucleotides for PCR assembly of the synthetic gene are designedby use of the publicly available software tool DNAWorks 3.1(http://helixweb.nih.gov/dnaworks/) and are assembled by PCR followingstandard protocols from the 18 oligos presented in Table 1 and as SEQ IDNo. 5 through SEQ ID No. 22 (Hoover D M, Lubkowski J. , DNAWorks: anautomated method for designing oligonucleotides for PCR-based genesynthesis, Nucleic Acids Res. 2002 May 15; 30(10):e43).

TABLE 1 Oligonucleotides used for assembly of the syntheticgene coding for the engineered VHH gene.1. CCATGGCAAGTTCAGCTGCAGGAAAGCGGTGGCGGCCTG (SEQ ID No. 5)2. AGACGCAGGCTGCCGCCAGGCTGGACCAGGCCGCCACCGC (SEQ ID No. 6)3. CGGCAGCCTGCGTCTGAGCTGTGCGGCCAGCGGCCGTACC (SEQ ID No. 7)4. AGGTGTAGCCGCTATGGTCGCTAAAGGTACGGCCGCTGGC (SEQ ID No. 8)5. ACCATAGCGGCTACACCTATACCATTGGCTGGTTTCGTCA (SEQ ID No. 9)6. TCACGTTCTTTTCCTGGCGCCTGACGAAACCAGCCAATGG (SEQ ID No. 10)7. CGCCAGGAAAAGAACGTGAATTTGTGGCGCGTATTTACTG (SEQ ID No. 11)8. ATAGGTATTGCCGCTGCTCCAGTAAATACGCGCCACAAAT (SEQ ID No. 12)9. GAGCAGCGGCAATACCTATTATGCGGATAGCGTGAAAGGC (SEQ ID No. 13)10. TGTCGCGGCTAATCGCGAAACGGCCTTTCACGCTATCCGC (SEQ ID No. 14)11. GCGATTAGCCGCGACATTGCCAAGAACACGGTAGATCTTA (SEQ ID No. 15)12. GGCTCCAGGTTGTTCATCGTAAGATCTACCGTGTTCTTGGC (SEQ ID No. 16)13. CGATGAACAACCTGGAGCCCGAAGACACAGCCGTGTATTA (SEQ ID No. 17)14. GCCATCCCGAGCCGCGCAATAATACACGGCTGTGTCTTCG (SEQ ID No. 18)15. GCGGCTCGGGATGGCATTCCGACCAGCCGTAGCGTGGAAA (SEQ ID No. 19)16. CCCTGGCCCCAGTAATTGTAGCTTTCCACGCTACGGCTGG (SEQ ID No. 20)17. CAATTACTGGGGCCAGGGCACCCAGGTGACCGTCAGCTCT (SEQ ID No. 21)18. GCGGCCGCAGAGCTGACGGTCACCTG (SEQ ID No. 22)

Briefly, equal volumes of oligonucleotide solutions (each at aconcentration of ˜1 mg/ml) are mixed together and diluted with water toa final concentration of ˜1 ng/μl for each oligonucleotide. Theoligonucleotide mixture is diluted 5-fold with the PCR solution. Thefinal concentrations of components are 0.2 ng/μl for eacholigonucleotide, 20 mM for Tris-HCl (pH 8.8), 10 mM for KCl, 10 mM for(NH₄)₂SO₄, 6 mM for MgSO₄, 0.1% (v/v) for Triton X-100, 0.1 mg/ml forbovine serum albumin, 0.2 mM for each dNTP and 2.5 U for Pfu polymerase.The PCR protocol for gene assembly begins with one 5 min denaturationstep of 95° C., during which the polymerase is added to avoid anypossible mispriming (‘hot start’ PCR). This step is followed by 25cycles of a denaturation temperature of 95° C. for 30 s, an annealingtemperature of 55° C. for 30 s and an extension temperature of 72° C.for 1.5 min. The last step in this protocol is an incubation cycle at72° C. for 10 min. For gene amplification, 1 μl of the mixture resultingfrom the gene assembly reaction are used as the template, with theoutermost oligonucleotides (SEQ ID No. 5 and SEQ ID No. 22) used asprimers. The PCR protocol for gene amplification is essentially the sameas that for gene assembly. The assembled synthetic VHH gene issubsequently cloned via the NcoI and NotI restriction sites in thevector pET27b (Novagen, http://www.merckbiosciences.co.uk/product/69863;http://www.novagen.com) and the sequence is verified by DNA sequencing.

PCR is then used to construct the randomized library. The template forthe first 2 PCR reactions is the cloned synthetic VHH gene as describedabove. The primer pairs used for the first two PCR reactions are asfollows: 3esynmu1 (gactccatgg caagtgcaac tgcaggaaag cggaggcggtctggttnnsc cannsnnsnn snnsggcagc ctgcgtctga gct (SEQ ID No. 23)) and3esynmu2 (catgagatct acggtgttct tggcg (SEQ ID No. 24)); 3esynmu3(catgagatct tacgatgnns nnsttgnnsn nsnnsnnsnn sgaagatacg gcggtgtatt attg(SEQ ID No. 25)) and 3esyn2 (aatagcggcc gcagagctca cggtcacc (SEQ ID No.26)). The resulting PCR products are digested with BglII, ligated, andthe ligation product is used as template for a PCR reaction with primers3esyn1 (acgtccatgg caagtgcaac tgcag (SEQ ID No. 27)) and 3esyn2(aatagcggcc gcagagctca cggtcacc (SEQ ID No. 26)). The NNS codons inprimers 3esynmu1 (SEQ ID No. 23) and 3esynmu3 (SEQ ID No. 25) introducethe randomized positions in the sequence. The codon NNS (IUPAC code,where S means C or G) is chosen which encodes all 20 naturally occurringamino acids, but avoids 2 out of 3 stop codons. FIG. 3 shows theschematic of the PCR reactions and ligation procedure. Horizontal arrowsindicate the positions and directions of the PCR primers, vertical linesindicate the positions of the NcoI, BglII and NotI sites, respectively(from left to right).

This randomized PCR product is subsequently cloned into the phagemidcloning vector pHEN1 (Hoogenboom H R, Griffiths A D, Johnson K S,Chiswell D J, Hudson P, Winter G. Multi-subunit proteins on the surfaceof filamentous phage: methodologies for displaying antibody (Fab) heavyand light chains. Nucleic Acids Res. 1991 Aug 11; 19(15):4133-7) inframe with the pelB secretion signal via the NcoI restriction site. TheNotI restriction site at the 3′ end of the gene inserts the VHH libraryin frame with the gene coding for the minor coat protein (protein III)of filamentous phage fd contained in the vector pHEN1. The engineeredsequence of the randomized VHH library insert is given as a nucleotidesequence in SEQ ID No. 28 and translated as an amino acid sequence inSEQ ID No. 29. The Letter X in SEQ ID No. 29 denotes the randomizedamino acid residues.

SEQ ID No. 26:   1ccatggcaag tgcaactgca ggaaagcgga ggcggtctgg ttnnsccann snnsnnsnns  61ggcagcctgc gtctgagctg cgcggcgtcc ggccgtacct ttagcgacca ttcgggctat 121acctatacca ttggctggtt ccgtcaggcg ccagggaaag aacgtgaatt tgtggcgcgt 181atttactgga gcagcggcaa tacctactat gcggatagcg tgaaaggccg ttttgcgatt 241agccgcgaca tcgccaagaa caccgtagat cttacgatgn nsnnsttgnn snnsnnsnns 301nnsgaagata cggcggtgta ttattgcgca gcgcgtgacg gcattccgac ctcccgtagc 361gtggaaagct acaattactg gggccagggc acccaggtga ccgtgagctc tgcggccgcSEQ ID No. 29:PWQVQLQESGGGLVXPXXXXGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMXXLXXXXXEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSAA

The ligation product is then transformed into Escherichia coli TG1, thenumber of obtained colonies is determined, and a number of selectedclones are controlled by restriction analysis and by DNA sequencing. Forthe following steps of the surface display library phage preparation,standard protocols are followed. Briefly, the ligation mixture istransformed into E. coli TG1 cells by electroporation. Subsequently,phage particles are rescued from E. coli TG1 cells with helper phageM13-KO7. Phage particles are then precipitated from culture supernatantwith PEG/NaCl in 2 steps, dissolved in water and used for selection bypanning or, alternatively, they are stored at minus 80° C.

EXAMPLE 3 Panning of the VHH-Phage Library on Human Serum Albumin (HSA)

3 panning rounds are performed according to standard protocols. (e.g.Phage display of peptides and antibodies: a laboratory manual, Kay etal., 1996, Academic Press, San Diego, Calif.) Briefly, the followingmethod is applied. Maxisorp 96-well plates (Nunc) are coated with HSA.200 μl of the following solution are added per well: 0.1M Na-carbonatebuffer, pH 9.6, with the following concentrations of HSA: 1^(st) panninground: 2 mg/ml HSA; 2nd panning round: 1 mg/ml HSA; 3^(rd) panninground: 1 mg/ml HSA Incubation is for 1 hour at 37° C., followed byblocking with 2% dry milk (M-PBS) with 200 μl per well for 1 hour atroom temperature. The surface display phage library is then allowed toreact with the bound HSA by adding 100 μl phage suspension and 100 μl 4%dry milk (M-PBS), followed by incubation for 45 minutes with shaking andfor 90 minutes without shaking at room temperature.

Unbound phage particles are washed away as follows. After the 1^(st)panning round: 10×300 μl T-PBS, 5×300 μl PBS; after the 2^(nd) panninground: 15×300 μl T-PBS, 10×300 μl PBS; after the 3^(rd) panning round:20×300 μl T-PBS, 20×300 μl PBS.

Elution of bound phage particles is performed by adding 200 μl per wellof 0.1 M glycine, pH 2.2, and incubation with shaking for 30 minutes atroom temperature. Subsequently, the phage suspension is neutralized byaddition of 60 μl 2M Tris-Base, followed by infection into E. coli TG1cells by mixing 10 ml exponentially growing culture with 0.5 ml elutedphage and incubation for 30 minutes at 37° C. Finally, infected bacteriaare plated on TYE medium with 1% glucose and 100 μg/ml ampicillin, andincubated at 30° C. overnight.

EXAMPLE 4 Cloning of Selected Clones of VHH Mutants Selected Against HSAfor Soluble Expression

Phagemid DNA from the phage selected through the 3 panning rounds isisolated with a midi-prep. DNA encoding mutated VHH domains regions isbatch-amplified by PCR and cloned NcoI-NotI into the vectorpNOTBAD/Myc-His, which is the E. coli expression vector pBAD/Myc-His(Invitrogen) with an inserted NotI restriction site to facilitatecloning. Ligated constructs are transformed into E. coli LMG194 cells(Invitrogen) with electroporation, and grown at 30° C. on TYE mediumwith 1% glucose and ampicillin overnight. Selected clones are inoculatedinto 200 μl 2×YT medium with ampicillin, grown overnight at 30° C., andinduced by adding L-arabinose to an end concentration of 0.1%. Afterexpression at 16° C. overnight, the cells are harvested bycentrifugation and treated with 100 μl Na-borate buffer, pH 8.0, at 4°C. overnight for preparation of periplasmic extracts. 50 μl of theperiplasmic extracts are used in ELISA (see below).

EXAMPLE 5 ELISA of VHH Mutants Selected Against HSA

The periplasmatic extracts of the VHH mutants selected for human serumalbumin-binding are tested in an ELISA with the following protocol:

Coating: Microtiter plate (NUNC, Maxisorp), 100 μl per well, 100 μgHSA/ml in PBS, overnight at 4° C.

Wash: 3× 200 μl PBS

Blocking: 1% Blocker Casein in PBS (Pierce), 1 h at RT

Wash: 3× 200 μl PBS

Periplasmic extract binding: 50 μl periplasmic extract (Example 4), 50μl PBS 0.05% Tween 20, at room temperature overnight Wash: 3× 200 μl PBS

1^(st) antibody: anti-His4 (Qiagen), 1:1000 in PBS 0.05% Tween 20, 90min at RT, 100 μl per well

Wash: 3× 200 μl PBS

2^(nd) antibody: goat anti mouse*HRP (SIGMA), 1:1000 in PBS 0.05%

Tween 20, 90 min at RT (room temperature), 100 μl per well

Wash: 3× 200 μl PBS

Detection: 3mg/ml OPD in Na-citrate/phosphate buffer, pH 4.5, 0.4 μl 30%H₂O₂

Stopping before background gets too high: 100 ml 3M H2SO4

Absorbance read: 492/620 nm

EXAMPLE 6 Example of a Library in Which Only One Loop is Randomized: theC″D Loop

The synthetic gene coding for the engineered VHH gene described above inexample 2 is used as a template for two PCR reactions in which thefollowing primer pairs are used: SEQ ID No. 30 (actgctcgag agacatcgccaagaacac; esynmu4) together with SEQ ID No. 26 (3esyn2) and SEQ ID No.31 (cacactcgag atcgcaaasn nsnnsnncac snnsnncgca tagtaggtat tgcc;3esynmu5) together with SEQ ID No. 27 (3esyn1). The resulting PCRproducts are digested with XhoI, ligated, and the ligation product isused as template for a PCR reaction with primers 3esyn1 (SEQ ID No. 27)and 3esyn2 (SEQ ID No. 26). The NNS codons in primers 3esynmu4 (SEQ IDNo. 30) and 3esynmu5 (SEQ ID No. 31) introduce the randomized positionsin the sequence similarily as described in example 2. FIG. 4 shows theschematic of the PCR reactions and ligation procedure. Horizontal arrowsindicate the positions and directions of the PCR primers, vertical linesindicate the positions of the NcoI, XhoI and NotI sites, respectively(from left to right).

This randomized PCR product is subsequently cloned into the phagemidcloning vector pHEN1 (Hoogenboom H R, Griffiths A D, Johnson K S,Chiswell D J, Hudson P, Winter G. Multi-subunit proteins on the surfaceof filamentous phage: methodologies for displaying antibody (Fab) heavyand light chains. Nucleic Acids Res. 1991 Aug 11; 19(15):4133-7) inframe with the pelB secretion signal and the minor coat protein (proteinIII) of filamentous phage fd contained in the vector pHEN1 as describedin example 2. The engineered sequence of the randomized VHH libraryinsert is given as a nucleotide sequence in SEQ ID No. 32 and translatedas an amino acid sequence in SEQ ID No. 33. The Letter X in SEQ ID No.33 denotes the randomized amino acid residues.

SEQ ID No. 32   1ccatggcaag ttcagctgca ggaaagcggt ggcggcctgg tccagcctgg cggcagcctg  61cgtctgagct gtgcggccag cggccgtacc tttagcgacc atagcggcta cacctatacc 121attggctggt ttcgtcaggc gccaggaaaa gaacgtgaat ttgtggcgcg tatttactgg 181agcagcggca atacctatta tgcgnnsnns gtgnnsnnsn nsttcgcgat ctcgagagac 241attgccaaga acacggtaga tcttacgatg aacaacctgg agcccgaaga cacagccgtg 301tattattgcg cggctcggga tggcattccg accagccgta gcgtggaaag ctacaattac 361tggggccagg gcacccaggt gaccgtcagc tctgcggccg c SEQ ID No. 33PWQVQLQESGGGLVQPGGSLRLSCAASGRTESDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYAXXVXXXFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSAA

The ligation product is then transformed into Escherichia coli TG1, thenumber of obtained colonies is determined, and a number of selectedclones are controlled by restriction analysis and by DNA sequencing. Forthe following steps of the surface display library phage preparation,standard protocols are followed as described in example 2. The steps ofpanning, selection and characterization of specifically binding clonesare performed essentially as described in examples 3, 4 and 5.

EXAMPLE 7 Example of a Library in Which Three Loops are Randomized: theAB, the EF and the C″D Loop

The library with randomized residues in the AB loop and in the EF loopas described in example 2 is used as a template for a PCR in which thesame primer pairs as in example 6 are used: SEQ ID No. 30 (esynmu4)together with SEQ ID No. 26 (3esyn2) and SEQ ID No. 31 (3esynmu5)together with SEQ ID No. 27 (3esyn1). The following steps for libraryconstruction, cloning, panning, selection and characterization ofspecifically binding clones are essentially as described in examples 2,3, 4 and 5.

EXAMPLE 8 Comparison of Variable Domain Libraries with Randomized AminoAcid Positions in One, Two and Three Structural Loops

The libraries are used in panning with various antigens.

hen-egg lysozyme as antigen:

3 panning rounds are performed. Maxisorp 96-well plates (Nunc) arecoated with hen egg lysozyme by adding 200 μl of the following solutionper well:

-   -   PBS, with the following concentrations of dissolved hen egg        lysozyme (HEL):    -   1st panning round: 2 mg/ml HEL    -   2nd panning round: 1 mg/ml HEL    -   3rd panning round: 1 mg/ml HEL

Incubation is for 1 hour at 37° C., followed by blocking with 2% drymilk (M-PBS) with 200 μl per well for 1 hour at room temperature.

The surface display phage library is then allowed to react with thebound hen egg lysozyme by adding 100 μl phage suspension and 100 μl 4%dry milk (M-PBS), followed by incubation for 45 minutes with shaking andfor 90 minutes without shaking at room temperature.

-   -   Unbound phage particles were washed away as follows:    -   1st panning round: 10×300 μl T-PBS, 5×300 μl PBS    -   2nd panning round: 15×300 μl T-PBS, 10×300 μl PBS    -   3rd panning round: 20×300 μl T-PBS, 20×300 μl PBS

Elution of bound phage particles is performed by adding 200 μl per wellof 0.1 M glycine, pH 2.2, and incubation with shaking for 30 minutes atroom temperature. Subsequently, the phage suspension is neutralized byaddition of 60 μl 2M Tris-Base, followed by infection into E. coli TG1cells by mixture of 10 ml exponentially growing culture with 0.5 mleluted phage and incubation for 30 minutes at 37° C. Finally, infectedbacteria are plated on TYE medium with 1% glucose and 100 μg/mlAmpicillin, and incubated at 30° C. overnight.

Human Serum Albumin as Antigen:

The libraries of examples 2, 6 and 7 are used in panning rounds asdescribed above. Specifically, the phage libraries are suspended inbinding buffer (PBS, 1% ovalbumin, 0.005% Tween 20) and panned againsthuman serum albumin immobilized directly on maxisorp plates (10micrograms/ml in PBS, overnight at 4° C.; plates are blocked withBlocker Casein (Pierce). After 2 hours, unbound phages are removed byrepetitive washing (PBS, 0.05% Tween 20) and bound phage are eluted with500 mM KCl, 10 mM HCl, pH 2.

After each HSA-specific panning round the resulting clones are selectedor tested for binding to TNF alpha. Selection and testing for TNF-alphaspecificty is performed as described in example 1 of WO2004/041862.

FcRn as Antigen:

The panning is performed as described in WO02060919, Example 6.2. Inshort, phage libraries are resuspended in 5 ml 20 mM MES, pH 6.0/5%skimmed milk/0.05% Tween 20 and added (100 micro-litre of 5×10¹²PFU/ml/well) to 20 wells of a Maxisorp immunoplate (Nunc) previouslycoated with 1 microgram of murine FcRn and blocked with 5% skimmed milk.After incubation for 2 h at 37° C., wells are washed 10-30 times with 20mM MES, pH 6.0/0.2% Tween 20/0.3 M NaCl and phage eluted by incubationin 100 microlitre PBS, pH 7.4/well for 30 min at 37° C. Phages are usedto reinfect exponentially growing E. coli TG1, as described in example3.

After each panning round on FcRn the resulting clones are selected ortested for binding to TNF alpha as described above.

Fc-Gamma Receptors as Antigens:

Panning against recombinant fusion proteins of Fc-gammaRI, Fc-gammaRIIAand Fc-gammaRIIA are performed as described in Berntzen et al (2006)Protein Eng Des Set 19:121-128 After each panning round on anFc-receptor the resulting clones are selected or tested for binding toTNF alpha as described above.

EXAMPLE 9

This example demonstrates the possibility to introduce new functions oradditional binding specificities into an antibody fragment. The moleculeused as a starting point for modification is the murine single chainantibody fragment sFv 26-10 (Huston et al. (1988) Proc Natl Acad SciUSA. 85:5879-5883). Five different libraries are constructed to modifydifferent structural loop sequences by random amino acid sequences:

Library 26-10-1: (SEQ ID NO. 34) EVQLQQSGPELVKPGASVRMSCKSSGYIFTDFYMNWVRQSHGKSLDYIGYISPYSGVTGYNQKF KGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAGSSGNKWAMDYWGHGASVTVSSGGGGSGGGG SGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLNWYLQKAGQSPKLLIYKVSN RFSGXXXXFSGSGSGTDFTLKISXXXXXXXGIYFCSQTTKVPPTFGGGTKLEIKR Library 26-10-2: (SEQ ID NO. 35) EVQLQQSGPELVKPGASVRMSCKSSGYIFTDFYMNWVRQSHGKSLDYIGYISPYSGVTGYNQKF KGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAGSSGNKWAMDYWGHGASVTVSSGGGGSGGGG SGGGGSDVVMTQTPLSLPXXXXXQASISCRSSQSLVHSNGNTYLNWYLQKAGQSPKLLIYKVSN RFSGXXXXFSGSGSGTDFTLKISXXXXXXXGIYFCSQTTHVPPTFGGGTKLEIKR Library 26-10-3: (SEQ ID NO. 36) EVQLQQSGPELVKPGASVRMSCKSSGYIFTDFYMNWVRQSHGKSLDYIGYISPYSGVTGYNQKF KGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAGSSGNKWAMDYWGHGASVTVSSGGGGSGGGG SGGGGSDVVMTQTPLSLPXXXXXQASISCRSSQSLVHSNGNTYLNWYLQKAGQSPKLLIYKVSN RFSGXVPDRFSGSGSGTDFTLKISXXXXXXXGIYFCSQTTHVPPTFGGGTKLEIKR Library 26-10-4: (SEQ ID NO. 37) EVQLQQSGPELVKPGASVRMSCKSSGYIFTDFYMNWVRQSHGKSLDYIGYISPYSGVTGYNQKF KGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAGSSGNKWAMDYWGHGASVTVSSGGGGSGGGG SGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLNWYLQKAGQSPKLLIYKVSN RFSGXXXXXXXFSGSGSGTDFTLKISXXXXXXXGIYFCSQTTHVPPTFGGGTKLEIKR Library 26-10-5: (SEQ ID NO. 38) EVQLQQSGPELVKPGASVRMSCKSSGYIFTDFYMNWVRQXXXXXXDYIGYISPYSGVTGYNQKF KGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAGSSGNKWAMDYWGHGASVTVSSGGGGSGGGG SGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLNWYLQXXXXXXKLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQTTHVPPTEGGGTKLEIKR The libraries are produced by reverse translation of the sequences, wherein the randomized amino acid positions are encoded by the nucleotide triplet NNK.  Library 26-10-1 gene:(SEQ ID NO. 39) GAAGTTCAGCTGCAGCAGTCTGGTCCGGAACTGGTTAAACCGGGTGCTTCTGTTCGTATGTCTTGCAAATCTTCTGGTTACATCTTCACCGACTTCTACATGAACTGGGTTCGTCAGTCTCACGGTAAATCTCTGCACTACATCGGTTACATCTCTCCGTACTCTGGTGTTACCGGTTACAACCAGAAATTCAAAGGTAAAGCTACCCTGACCGTTGACAAATCTTCTTCTACCGCTTACATGGAACTGCGTTCTCTGACCTCTGAAGACTCTGCTGTTTACTACTGCGCTGGTTCTTCTGGTAACAAATGGGCTATGGACTACTGGGGTCACGGTGCTTCTGTTACCGTTTCTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGACGTTGTTATGACCCAGACCCCGCTGTCTCTGCCGGTTTCTCTGGGTGACCAGGCTTCTATCTCTTGCCGTTCTTCTCAGTCTCTGGTTCACTCTAACGGTAACACCTACCTGAACTGGTACCTGCAGAAAGCTGGTCAGTCTCCGAAACTGCTGATCTACAAAGTTTCTAACCGTTTCTCTGGTNNKNNKNNKNNKTTCTCTGGTTCTGGTTCTGGTACCGACTTCACCCTGAAAATCTCTNNKNNKNNKNNKNNKNNKNNKGGTATCTACTTCTGCTCTCAGACCACCCACGTTCCGCCGACCTTCGGTGGTGGTACCAAACTGGAAATCAAACGT Library 26-10-2 gene:(SEQ ID NO. 40) GAAGTTCAGCTGCAGCAGTCTGGTCCGGAACTGGTTAAACCGGGTGCTTCTGTTCGTATGTCTTGCAAATCTTCTGGTTACATCTTCACCGACTTCTACATGAACTGGGTTCGTCAGTCTCACGGTAAATCTCTGGACTACATCGGTTACATCTCTCCGTACTCTGGTGTTACCGGTTACAACCAGAAATTCAAAGGTAAAGCTACCCTGACCGTTGACAAATCTTCTTCTACCGCTTACATGGAACTGCGTTCTCTGACCTCTGAAGACTCTGCTGTTTACTACTGCGCTGGTTCTTCTGGTAACAAATGGGCTATGGACTACTGGGGTCACGGTGCTTCTGTTACCGTTTCTTCTGGTGGTGCTGGTTCTGCTGCTGCTGGTTCTGGTGGTGGTGGTTCTGACGTTGTTATGACCCAGACCCCGCTGTCTCTGCCGNNKNNKNNKNNKNNKCAGGCTTCTATCTCTTGCCGTTCTTCTCAGTCTCTGGTTCACTCTAACGGTAACACCTACCTGAACTGGTACCTGCAGAAAGCTGGTCAGTCTCCGAAACTGCTGATCTACAAAGTTTCTAACCGTTTCTCTGGTNNKNNKNNKNNKTTCTCTGGTTCTGGTTCTGGTACCGACTTCACCCTGAAAATCTCTNNKNNKNNKNNKNNKNNKNNKGGTATCTACTTCTGCTCTCAGACCACCCACGTTCCGCCGACCTTCGGTGGTGGTACCAAACTGGAAATCAAACGT Library 26-10-3 gene:(SEQ ID NO. 41) GAAGTTCAGCTGCACCAGTCTGGTCCGGAACTGGTTAAACCGGGTGCTTCTGTTCGTATGTCTT GCAAATCTTCTGGTTACATCTTCACCGACTTCTACATGAACTGGGTTCGTCAGTCTCACGGTAA ATCTCTGGACTACATCGGTTACATCTCTCCGTACTCTGGTGTTACCGGTTACAACCAGAAATTC AAAGGTAAAGCTACCCTGACCGTTGACAAATCTTCTTCTACCGCTTACATGGAACTGCGTTCTC TGACCTCTGAAGACTCTGCTGTTTACTACTGCGCTGGTTCTTCTGGTAACAAATGGGCTATGGA CTACTGGGGTCACGGTGCTTCTGTTACCGTTTCTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT TCTGGTGGTGGTGGTTCTGACGTTGTTATGACCCAGACCCCGCTGTCTCTGCCGNNKNNKNNKN NKNNKCAGGCTTCTATCTCTTGCCGTTCTTCTCAGTCTCTGGTTCACTCTAACGGTAACACCTA CCTGAACTGGTACCTGCAGAAAGCTGGTCAGTCTCCGAAACTGCTGATCTACAAAGTTTCTAAC CGTTTCTCTGGTGTTCCGGACCGTTTCTCTGGTTCTGGTTCTGGTACCGACTTCACCCTGAAAA TCTCTNNKNNKNNKNNKNNKNNKNNKGGTATCTACTTCTGCTCTCAGACCACCCACGTTCCGCC GACCTTCGGTGGTGGTACCAAACTGGAAATCAAACGT  Library 26-10-4 gene:(SEQ ID NO. 42) GAAGTTCAGCTGCAGCAGTCTGGTCCGGAACTGGTTAAACCGGGTGCTTCTGTTCGTATGTCTT GCAAATCTTCTGGTTACATCTTCACCGACTTCTACATGAACTGGGTTCGTCAGTCTCACGGTAA ATCTCTGGACTACATCGGTTACATCTCTCCGTACTCTGGTGTTACCGGTTACAACCAGAAATTC AAAGGTAAAGCTACCCTGACCGTTGACAAATCTTCTTCTACCGCTTACATGGAACTGCGTTCTC TGACCTCTGAAGACTCTGCTGTTTACTACTGCGCTGGTTCTTCTGGTAACAAATGGGCTATGGA CTACTGGGGTCACGGTGCTTCTGTTACCGTTTCTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT TCTGGTGGTGGTGGTTCTGACGTTGTTATGACCCAGACCCCGCTGTCTCTGCCGGTTTCTCTGG GTGACCAGGCTTCTATCTCTTGCCGTTCTTCTCAGTCTCTGGTTCACTCTAACGGTAACACCTA CCTGAACTGGTACCTGCAGAAAGCTGGTCAGTCTCCGAAACTGCTGATCTACAAAGTTTCTAAC CGTTTCTCTGGTNNKNNKNNKNNKNNKNNKNNKTTCTCTGGTTCTGGTTCTGGTACCGACTTCA CCCTGAAAATCTCTNNKNNKNNKNNKNNKNNKNNKGGTATCTACTTCTGCTCTCAGACCACCCA CGTTCCGCCGACCTTCGGTGGTGGTACCAAACTGGAAATCAAACGT  Library 26-10-5 gene:(SEQ ID NO. 43) GAAGTTCAGCTGCAGCAGTCTGGTCCGGAACTGGTTAAACCGGGTGCTTCTGTTCGTATGTCTT GCAAATCTTCTGGTTACATCTTCACCGACTTCTACATGAACTGGGTTCGTCAGNNKNNKNNKNN KNNKNNKGACTACATCGGTTACATCTCTCCGTACTCTGGTGTTACCGGTTACAACCAGAAATTC AAAGGTAAAGCTACCCTGACCGTTGACAAATCTTCTTCTACCGCTTACATGGAACTGCGTTCTC TGACCTCTGAAGACTCTGCTGTTTACTACTGCGCTGGTTCTTCTGGTAACAAATGGGCTATGGA CTACTGGGGTCACGGTGCTTCTGTTACCGTTTCTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT TCTGGTGGTGGTGGTTCTGACGTTGTTATGACCCAGACCCCGCTGTCTCTGCCGGTTTCTCTGG GTGACCAGGCTTCTATCTCTTGCCGTTCTTCTCAGTCTCTGGTTCACTCTAACGGTAACACCTA CCTGAACTGGTACCTGCAGNNKNNKNNKNNKNNKNNKAAACTGCTGATCTACAAAGTTTCTAAC CGTTTCTCTGGTGTTCCGGACCGTTTCTCTGCTTCTGGTTCTGGTACCGACTTCACCCTGAAAA TCTCTCGTGTTGAAGCTGAAGACCTGGGTATCTACTTCTGCTCTCAGACCACCCACGTTCCGCC GACCTTCGGTGGTGGTACCAAACTGGAAATCAAACGT 

The respective genes are assembled with chemically synthesizedoligonucleotides and cloned in a phage display vector for phage surfacedisplay as described above with all necessary adaptions to achievein-frame translation of a leader peptide, the sFv-variant and the coatprotein III of the phage.

The phage displayed displayed libraries are selected for binding tohuman serum albumine, lysozyme, FcRn and Fc gamma receptors as describedin example 8.

Alternatively the genes are ligated with the vector pRDV analog to themethod described in Binz et al. (2005) Nat Biotechnol. 22:575-582 andpanned for human serum albumine, Fc-receptors and lysozyme according tothe methodology described in Schaffitzel et al. (1999) J Immunol Methods231:119-135.

EXAMPLE 10

A human antibody, 2F5, specific for the HIV-peptide ELDKWA is used as ascaffold for randomization of structural loops and expressed as scFv.Expression and Phage display vector construction is performed asdescribed in example 9. Phage selection is performed accordingly.

The wild type scFv sequence and the respective libraries are shown inFIGS. 5 to 8.

EXAMPLE 11 Fab Library, in which Structural Loops of a Constant Domainare Randomized

For the selection of specifically binding molecules from libraries ofimmunoglobulin domains, in which residues that are located in structuralloops have been randomized, various formats can be applied. Singledomains, such as VL, VH, CH1, CH2, CH3, CH4 or CL domains can be used,Fc fragments which consist of CH2 and CH3 domains (including or notincluding the hinge region or parts thereof) can be used, Fv orsingle-chain Fv fragments can be used, whole antibodies can be used orother combinations of immunoglobulin domains can be used. One formatthat is of particular interest for the selection of specifically bindingmolecules is the Fab fragment, which is a heterodimer of two chains,namely the VL-CL part and the VH-CH1 part of the antibody. Fab fragmentshave been known for a long time, and can be produced by proteolyticcleavage of an IgG with the protease papain, and they can also beproduced recombinantly in a wide range of different expression systems,such as for example Escherichia coli, Saccharomyces cerevisiae, Pichiapastoris, insect cells or mammalian cells.

It is well known in the art that surface display systems, such as phagedisplay, yeast display, and other systems such as ribosome display etc.can be used for the enrichment and selection of specifically bindingmolecules such as Fab fragments from large libraries (See for exampleHoogenboom H R, Griffiths A D, Johnson K S, Chiswell D J, Hudson P,Winter G.

Multi-subunit proteins on the surface of filamentous phage:methodologies for displaying antibody (Fab) heavy and light chains.Nucleic Acids Res. 1991 Aug 11; 19(15):4133-7.; Kang A S, Barbas C F,Janda K D, Benkovic S J, Lerner R A. Linkage of recognition andreplication functions by assembling combinatorial antibody Fab librariesalong phage surfaces. Proc Natl Acad Sci USA. 1991 May 15;88(10):4363-6.; Kang X, Yang B A, Hu Y, Zhao H, Xiong W, Yang Y, Si B,Zhu Q. Human neutralizing Fab molecules against severe acute respiratorysyndrome coronavirus generated by phage display. Clin Vaccine Immunol.2006 Aug;13(8):953-7.; Weaver-Feldhaus J M, Lou J, Coleman J R, Siegel RW, Marks J D, Feldhaus M J. Yeast mating for combinatorial Fab librarygeneration and surface display. FEBS Lett. 2004 Apr 23;564(1-2):24-34.).

As an example, if the phage display system is applied for the display ofFab fragments (e.g. libraries of Fab fragments), one chain of the Fabfragment, e.g. the VH-CH1 chain, can be expressed as a fusion proteinwith e.g. protein III of phage M13, thereby leading to the display ofthis chain on the phage surface, while the other chain, VL-CL, isexpressed in soluble form and forms the natural heterodimer with thesurface anchored VH-CH1 chain. In a typical Fab surface display library,different VH and VL sequences are present, which can originate from adonor, typically a mouse or a human, but diversity can also be generatedby in vitro methods such as site directed mutagenesis. Different bindingsites are thereby generated, and by the use of a suitable display orother enrichment or selection method specifically binding clones can beisolated from such libraries that bind to their binding partner via thebinding site that is formed by VH and VL.

Fab fragments can be generated that bind to one target via their naturalbinding site formed by VH and VL, and to another (or a second time tothe same) target via binding sites formed by their structural loops. Inorder to obtain such engineered Fab fragments, libraries of Fabs arefirst generated in which residues in the structural loops are replacedby randomized sequences. Insertions of additional residues can also bemade. Structural loops that can be engineered by this approach can beeither C-terminal loops (the “bottom” loops) of VH or VL, or N-terminal(“top” loops) or C-terminal (“bottom” loops) of the CH1 or of the CLdomains. Different combinations of domains with engineered structuralloops at any of these positions are also possible. One format that canbe used to select specifically binding domains is as single domains, asFv or single-chain Fv fragments, or, as described in detail below, asFab fragments.

The genes coding for VH-CH1 and VL-CL respectively of the antibody 4D5(Cho H S, Mason K, Ramyar K X, Stanley A M, Gabelli S B, Denney D W Jr,Leahy D J. Structure of the extracellular region of HER2 alone and incomplex with the Herceptin Fab. Nature. 2003 Feb 13; 421(6924):756-60.),which binds to human Her2 are used for this example. A synthetic gene isconstructed which consists of the 4D5 VL-CL encoding part of the gene,flanked at its 5′ end by an NcoI site for in-frame insertion in the pelBsignal sequence contained in the phagemid vector pHEN1 (Hoogenboom H R,Griffiths A D, Johnson K S, Chiswell D J, Hudson P, Winter G.Multi-subunit proteins on the surface of filamentous phage:methodologies for displaying antibody (Fab) heavy and light chains.Nucleic Acids Res. 1991 Aug 11; 19(15):4133-7) followed by a stop codon.At the transit of the sequence from VL into CL, a unique BsiWIrestriction site is included, which is used later on for replacement ofthe wildtype CL sequence against library inserts containing randomizedstructural loops in combination with a unique AscI restriction site thatis located downstream of the stop codon of the VL-CL encoding gene. Itfollows a sequence which contains a ribomsome binding site(Shine-Dalgarno sequence) taken from Carter et al. (Carter P, Kelley RF, Rodrigues M L, Snedecor B, Covarrubias M, Velligan M D, Wong W L,Rowland A M, Kotts C E, Carver M E, et al. High level Escherichia coliexpression and production of a bivalent humanized antibody fragment.Biotechnology (NY). 1992 Feb;10(2):163-7), followed by a gene segmentencoding the heat stable enterotoxin II (stII) signal sequence fused inframe to the VH-CH1 encoding part of the antibody 4D5 followed by a NotIsite for in-frame insertion in the vector pHEN1. This dicistronicconstruct leads to the expression of VL-CL (the protein sequence ofwhich is given in SEQ ID No. 44) on the one hand an on the other hand ofVH-CH1 fused to protein III of phage M13 (the protein sequence of whichis given in SEQ ID No. 45), which is encoded by pHEN1. The completesequence of the 4D5 Fab display vector as described here is given as anucleotide sequence in SEQ ID No. 46.

For construction of a library of CL domains in which residues in thestructural loops are randomized, a synthetic gene is made which encodesa human kappa constant domain (CL) in which certain codons are replacedby a degenerated codon such as for example NNB (IUPAC code, where Nstands for C, G, T and A; B stands for T, C and G). It is also possibleto insert additional residues into the sequence. In this example, 3, 4or 5 residues respectively are inserted between residues 127 and 128,and residues 182-185 and 187-189 are randomized (Kabat numbering). Thesequences of the resulting genes are given as nucleotide sequences inSEQ ID No. 47, 48, and 49 (3, 4 or 5 insertions respectively betweenresidues 127 and 128) and as amino acid sequences in SEQ ID No. 50, 51and 52 (the letter X stands for any of the 20 naturally encoded aminoacids). The nucleotide sequences include the BsiWI site on the the 5′end and the AscI site at the 3′ end for cloning into SEQ ID No. 46.

To construct the phage display library, the Fab 4D5 display vector (SEQID No. 46) is cleaved with the restriction enzymes BsiWI and AscI, andthe large fragment is prepared by preparative agarose gelelectrophoresis. The small fragment, corresponding to the gene encodingthe wildtype CL is removed. The mixture of library inserts as describedabove (SEQ ID Nos. 47-49) and likewise cleaved with BsiWI and AscI andligated to the purified vector fragment. The ligation mixture istransformed into a suitable E. coli strain such as TG1 by e.g.electroporation, and a large number of independent colonies aregenerated (e.g. 10exp8, 10exp9 or more). The transformed bacteria arepooled and infected with helper phage (such as e.g. M13K07) for rescueof phage particles. The phage particles are produced by standardprocedures and used for panning of the library.

Panning of the library against a given target yields Fab fragments thatnot only bind to Her2 (due to binding site formed by VH and VL of theantibody 4D5) but also to the target against which they were selected.

In this example, the design, preparation and use of a Fab displaylibrary, in which structural loops of the CL domain are modified, isdescribed. In an analogous way, libraries with randomization in thestructural loops of other domains, such as the CH1, the VH or the VLdomain can be prepared and used.

Sequences:

SEQ ID No. 44       5        10         15       20        25         30  1 M K Y L L P T A A A G L L L L A A Q P A M A D I Q M T Q S P  31S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q  61K P G K A P K L L I Y S A S F L Y S G V P S R F S G S R S G  91T D F T L T I S S L Q P E D T A T Y Y C Q Q H Y T T P P T F 121G Q G T K V E I K R T V A A P S V F I F P P S D E Q L K S G 151T A S V V C L L N N E Y P R E A K V Q W K V D N A L Q S G N 181S Q E S V T E Q D S K D S T Y S L S S T L T L S K A D Y E K  211H K V Y A C E V T H Q G L S S P V T K S F N R G E C  /// SEQ ID No. 45      5        10         15       20        25         30   1M K K N I A F L L A S M F V F S I A T N A Y A E V Q L V E S  31G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V  61R Q A P G K G L E W V A R I Y P T N G Y T R Y A D S V K G R  91F T I S A D T S K N T A Y L Q M N S L R A E D T A V Y Y C S 121R W G G D G F Y A M D Y W G Q G T L V T V S S A S T K G P S 151V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V 181T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L S S 211V V T V P S S S L G T Q T Y I C N V N H K P S N T K V D K K 241V E P K S C A A A E Q K L I S E E D L N G A A @ T V E S C L 271A K P H T E N S F T N V W K D D K T L D R Y A N Y E G C L W 301N A T G V V V C T G D E T Q C Y G T W V P I G L A I P E N E 331G G G S E G G G S E G G G S E G G G T K P P E Y G D T P I P 361G Y T Y I N P L D G T Y P P G T E Q N P A N P N P S L E E S 391Q P L N T E M F Q N N R E R N R Q G A L T V Y T G T V T Q G 421T D P V K T Y Y Q Y T P V S S K A M Y D A Y W N G K F R D C 451A F H S G F N E D P F V C E Y Q G Q S S D L P Q P P V N A G 481G G S G G G S G G G S E G G G S E G G G S E G G G S E G G G 511S G G G S G S G D F D Y E K M A N A N K G A M T E N A D E N 541A L Q S D A K G K L D S V A T D Y G A A I D G F I G D V S G 571L A N G N G A T G D F A G S N S Q M A Q V G D G D N S P L M 601N N F R Q Y L P S L P Q S V E C R P Y V E G A G K P Y E F S 631I D C D K I N L E R G V F A F L L Y V A T F M Y V E S T F A 661N I L H K E S SEQ ID No. 46    1gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt   61cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt  121tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat  181aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt  241ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg  301ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga  361tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc  421tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac  481actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg  541gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca  601acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg  661gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg  721acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg  781gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag  841ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg  901gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct  961cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1021agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1081catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1141tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1201cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1261gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1321taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 1381ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1441tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1501ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1561cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1621agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1681gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1741atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1801gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1861gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1921ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1981cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2041cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2101acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2161cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2221accatgatta cgccaagctt gcatgcaaat tctatttcaa ggagacagtc ataatgaaat 2281acctattgcc tacggcagcc gctggattgt tattactcgc ggcccagccg gccatggccg 2341atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat agggtcacca 2401tcacctgccg tgccagtcag gatgtgaata ctgctgtagc ctggtatcaa cagaaaccag 2461gaaaagctcc gaaactactg atttactcgg catccttcct ctactctgga gtcccttctc 2521gcttctctgg atccagatct gggacggatt tcactctgac catcagcagt ctgcagccgg 2581aagacttcgc aacttattac tgtcagcaac attatactac tcctcccacg ttcggacagg 2641gtaccaaggt ggagatcaaa cgtacggtgg cggcgccatc tgtcttcatc ttcccgccat 2701ctgatgagca gcttaagtct ggaactgcct ctgttgtgtg cctgctgaat aacttctatc 2761ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt aactcccagg 2821agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc accctgacgc 2881tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc catcagggcc 2941tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttaataaggc gcgccgctga 3001tcctctacgc cggacgcatc gtggccctag tacgcaagtt cacgtaaaaa gggtatctag 3061aggttgaggt gattttatga aaaagaatat cgcatttctt cttgcatcta tgttcgtttt 3121ttctattgct acaaatgcat acgctgaggt tcaactagtg gagtctggcg gtggcctggt 3181gcagccaggg ggctcactcc gtttgtcctg tgcagcttct ggcttcaaca ttaaagacac 3241ctatatacac tgggtgcgtc aggccccggg taagggcctg gaatgggttg caaggattta 3301tcctacgaat ggttatacta gatatgccga tagcgtcaag ggccgtttca ctataagcgc 3361agacacatcc aaaaacacag cctacctgca gatgaacagc ctgcgtgctg aggacactgc 3421cgtctattat tgttctagat ggggagggga cggcttctat gctatggact actggggtca 3481aggaaccctg gtcaccgtct cctcggcgtc gaccaagggc ccatcggtct tccccctggc 3541accctcctcc aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta 3601cttccccgaa ccggtgacgg tgtcgtggaa ctcaggtgcc ctgaccagcg gcgtgcacac 3661cttcccggct gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc 3721ctccagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac 3781caaggtggac aagaaagttg agcccaaatc ttgtgcggcc gcagaacaaa aactcatctc 3841agaagaggat ctgaatgggg ccgcatagac tgttgaaagt tgtttagcaa aacctcatac 3901agaaaattca tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta 3961tgagggctgt ctgtggaatg ctacaggcgt tgtggtttgt actggtgacg aaactcagtg 4021ttacggtaca tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga 4081gggtggcggt tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg 4141tgatacacct attccgggct atacttatat caaccctctc gacggcactt atccgcctgg 4201tactgagcaa aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac 4261tttcatgttt cagaataata ggttccgaaa taggcagggt gcattaactg tttatacggg 4321cactgttact caaggcactg accccgttaa aacttattac cagtacactc ctgtatcatc 4381aaaagccatg tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg 4441ctttaatgag gatccattcg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc 4501tcctgtcaat gctggcggcg gctctggtgg tggttctggt ggcggctctg agggtggcgg 4561ctctgagggt ggcggttctg agggtggcgg ctctgagggt ggcggttccg gtggcggctc 4621cggttccggt gattttgatt atgaaaaaat ggcaaacgct aataaggggg ctatgaccga 4681aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac 4741tgattacggt gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa 4801tggtgctact ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga 4861taattcacct ttaatgaata atttccgtca atatttacct tctttgcctc agtcggttga 4921atgtcgccct tatgtctttg gcgctggtaa accatatgaa ttttctattg attgtgacaa 4981aataaactta ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt 5041attttcgacg tttgctaaca tactgcataa ggagtcttaa taagaattca ctggccgtcg 5101ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 5161atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 5221agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 5281gcggtatttc acaccgcacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa 5341gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 5401ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 5461ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 5521aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 5581gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 5641cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct 5701attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 5761cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 5821agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 5881ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 5941catcaccgaa acgcgcga SEQ ID No. 47   1cgtacggtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gcttaagtct  61nnbnnbnnbg gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc 121aaagtacagt ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca 181gagcaggaca gcaaggacag cacctacagc ctcagcagca ccctgacgct gnnbnnbnnb 241nnbtacnnbn nbnnbaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc 301gtcacaaaga gcttcaacag gggagagtgt taataaggcg cgcc SEQ ID No. 48   1cgtacggtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gcttaagtct  61nnbnnbnnbn nbggaactgc ctctgttgtg tgcctgctga ataacttcta tcccagagag 121gccaaagtac agtggaaggt ggataacgcc ctccaatcgg gtaactccca ggagagtgtc 181acagagcagg acagcaagga cagcacctac agcctcagca gcaccctgac gctgnnbnnb 241nnbnnbtacn nbnnbnnbaa agtctacgcc tgcgaagtca cccatcaggg cctgagctcg 301cccgtcacaa agagcttcaa caggggagag tgttaataag gcgcgcc SEQ ID No. 49   1cgtacggtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gcttaagtct  61nnbnnbnnbn nbnnbggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga 121gaggccaaag tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt 181gtcacagagc aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgnnb 241nnbnnbnnbt acnnbnnbnn baaagtctac gcctgcgaag tcacccatca gggcctgagc 301tcgcccgtca caaagagctt caacagggga gagtgttaat aaggcgcgcc  SEQ ID No. 50      5        10         15       20        25         30  1R T V A A P S V F I F P P S D E Q L K S X X X G T A S V V C 31L L N N F Y P R E A K V Q W K V D N A L Q S G N S Q E S V T 61E Q D S K D S T Y S L S S T L T L X X X X Y X X X K V Y A C 91E V T H Q G L S S P V T K S F N R G E C SEQ ID No. 51      5        10         15       20        25         30  1R T V A A P S V F I F P P S D E Q L K S X X X X G T A S V V 31C L L N N F Y P R E A K V Q W K V D N A L Q S G N S Q E S V 61T E Q D S K D S T Y S L S S T I T L X X X X Y X X X K V Y A 91C E V T H Q G L S S P V T K S F N R G E C SEQ ID No. 52      5        10         15       20        25         30  1R T V A A P S V F I F P P S D E Q L K S X X X X X G T A S V 31V C L L N N F Y P R E A K V Q W K V D N A L Q S G N S Q E S 61V T E Q D S K D S T Y S L S S T L T L X X X X Y X X X K V Y 91A C E V T H Q G L S S P V T K S F N R G E C

FIG. 1: Sequence alignment of the amino acid sequences of SEQ ID No. 1and SEQ ID No. 2.

FIG. 2: Nucleotide sequence encoding the synthetic gene coding for theanti-TNF-alpha camel VHH domain, as well as the translation in aminoacid one-letter code. Restriction sites used for cloning are underlinedin the nucleotide sequence. Amino acid residues to be randomized in thelibrary are underlined in the amino acid sequence. (Inserted amino acidsare not given in this sequence.)

FIG. 3 shows the schematic of the PCR reactions and ligation procedure.Horizontal arrows indicate the positions and directions of the PCRprimers, vertical lines indicate the positions of the NcoI, BglII andNotI sites, respectively (from left to right).

FIG. 4: Schematic of the PCR reactions used to construct the library asdescribed in example 6.

FIG. 5: scFv 2F5 wt synthetic gene library 1 (VH-Linker-VL) andtranslation, with two structural loops randomized

FIG. 6: scFv 2F5 wt synthetic gene library 2 (VH-Linker-VL) andtranslation, with two structural loops randomized

FIG. 7: scFv 2F5 wt synthetic gene library 3 (VH-Linker-VL) andtranslation, with three structural loops randomized

FIG. 8: scFv 2F5 wild-type synthetic gene with translation(VH-Linker-VL)

1. A polypeptide scaffold comprising an immunoglobulin fold of a humanantibody variable domain, wherein structural loops A-B, C-C′, C″-D andE-F of said human antibody variable domain do not comprise a CDR loop,wherein at least two of said structural loops A-B, C-C′, C″-D and E-F(i) comprise at least one amino acid selected from the group consistingof tryptophan, tyrosine, phenylalanine, histidine, isoleucine, serine,methionine, alanine and asparagine, and (ii) form a solvent accessiblesurface.
 2. The polypeptide scaffold of claim 1, wherein said humanantibody variable domain is selected from the group consisting of aVariable Heavy (VH) domain, a Variable kappa (Vκ) domain, a Variablelambda (Vλ) domain.
 3. The polypeptide scaffold of claim 1, wherein saidat least two structural loops of said human antibody variable domaincomprises at least one tyrosine in any one of the positions 8 to 20,amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101,and/or at least one tryptophan in any one of the positions 8 to 20,amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101,and/or at least one histidine in any one of the positions 8 to 20, aminoacids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101, and/orat least one asparagine in any one of the positions 8 to 20, amino acids44 to 50, amino acids 67 to 76 and amino acids 89 to 101, and/or atleast one methionine in any one of the positions 8 to 20, amino acids 44to 50, amino acids 67 to 76 and amino acids 89 to 101, and/or at leastone serine in any one of the positions 8 to 20, amino acids 44 to 50,amino acids 67 to 76 and amino acids 89 to 101, and/or at least oneisoleucine in any one of the positions 6 to 20, amino acids 44 to 52,amino acids 67 to 76 and amino acids 92 to 101 and/or at least onephenylalanine in any one of the positions 8 to 20, amino acids 44 to 50,amino acids 67 to 76 and amino acids 89 to 101, wherein the numbering isaccording to the ImMunoGeneTics numbering system (IMGT).
 4. Thepolypeptide scaffold of claim 1, wherein said at least two structuralloops of said human antibody variable domain form a solvent accessiblesurface within one or more of (i) amino acids 8 to 20, (ii) amino acids44 to 50, (iii) amino acids 67 to 76 and (iv) amino acids 89 to 101,wherein the numbering is according to the ImMunoGeneTics numberingsystem (IMGT)¹.
 5. The polypeptide scaffold of claim 1, wherein saidpolypeptide scaffold or fragment thereof, is further combined with oneor more of said polypeptide scaffolds or fragment thereof, or with oneor more polypeptides, or fragment thereof, to obtain a combinationpolypeptide.
 6. The polypeptide scaffold of claim 5, wherein saidcombination polypeptide is a fusion protein further comprising one ormore of the group consisting of an immunoglobulin, a ligand, an enzyme,and a toxin.
 7. The polypeptide scaffold of claim 1, wherein each ofsaid structural loops A-B, C-C′, C″-D and E-F comprises a solventaccessible surface.
 8. A kit comprising the polypeptide scaffold ofclaim
 1. 9. A polypeptide scaffold comprising an immunoglobulin fold ofa human antibody Constant domain, wherein the structural loops of saidhuman antibody constant domain are connected by beta strands, wherein atleast two of said structural loops (i) comprise at least one amino acidselected from the group consisting of tryptophan, tyrosine,phenylalanine, histidine, isoleucine, serine, methionine, alanine andasparagine, and (ii) form a solvent accessible surface.
 10. Thepolypeptide scaffold of claim 9, wherein said human antibody constantdomain is selected from the group consisting of a CH1 domain, a CH2domain and a CH3 domain.
 11. The polypeptide scaffold of claim 9,wherein said at least two structural loops of said human antibodyconstant domain comprises at least one tyrosine in any one of thepositions 12 to 17, 45 to 50, 69 to 75 and 93 to 98, and/or at least onetryptophan in any one of the positions 12 to 17, 45 to 50, 69, 71 to 75,93 to 94 and 96 to 98, and/or at least one histidine in any one of thepositions 12 to 17, 46, 47, 49, 50, 69 to 74 and 93 to 98, and/or atleast one asparagine in any one of the positions 12 to 17, 45 to 47, 49,50, 70 to 73, 75, 94 to 96 and 98, and/or at least one methionine in anyone of the positions 12 to 17, 46 to 50, 69 to 71, 73 to 75, 93, 95, 96and 98, and/or at least one serine in any one of the positions 13, 71,75, 94, 95 and 98, and/or at least one isoleucine in any one of thepositions 12, 14 to 17, 45 to 50, 69, 70, 72 to 75, 93 and 96 to 98,and/or at least one phenylalanine in any one of the positions 15, 46,48, 70 to 73, 75, 93, 95 and 98, wherein the numbering is according tothe ImMunoGeneTics numbering system (IMGT).
 12. The polypeptide scaffoldof claim 9, wherein said at least two structural loops of said humanantibody constant domain comprise at least one solvent accessiblesurface within one or more of (i) amino acids 7 to 21, (ii) amino acids25 to 39, (iii) amino acids 41 to 81, (iv) amino acids 83 to 85, (v)amino acids 89 to 103 and (vi) amino acids 106 to 117, wherein thenumbering is according to the ImMunoGeneTics numbering system (IMGT).13. The polypeptide scaffold of claim 9, wherein said polypeptidescaffold or fragment thereof, is further combined with one or more saidpolypeptide scaffold or fragments thereof, or with one or morepolypeptides, or fragments thereof, to obtain a combination polypeptide.14. The polypeptide scaffold of claim 13, wherein said combinationpolypeptide is a fusion protein further comprising one or more of thegroup consisting of another immunoglobulin, a ligand, an enzyme, and atoxin.
 15. The polypeptide scaffold of claim 9, wherein three of said atleast two structural loops of said human antibody constant domain form asolvent accessible surface.
 16. A kit comprising the polypeptidescaffold of claim 9.